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. 2022 Sep 2;5(10):932–944. doi: 10.1021/acsptsci.2c00113

Novel and Structurally Diversified Bacterial DNA Gyrase Inhibitors Discovered through a Fluorescence-Based High-Throughput Screening Assay

Eddy E Alfonso †,‡, Zifang Deng †,‡, Daniel Boaretto †,‡, Becky L Hood §, Stefan Vasile §, Layton H Smith §, Jeremy W Chambers †,⊥, Prem Chapagain †,#, Fenfei Leng †,‡,*
PMCID: PMC9578135  PMID: 36268121

Abstract

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Bacterial DNA gyrase, a type IIA DNA topoisomerase that plays an essential role in bacterial DNA replication and transcription, is a clinically validated target for discovering and developing new antibiotics. In this article, based on a supercoiling-dependent fluorescence quenching (SDFQ) method, we developed a high-throughput screening (HTS) assay to identify inhibitors targeting bacterial DNA gyrase and screened the National Institutes of Health’s Molecular Libraries Small Molecule Repository library containing 370,620 compounds in which 2891 potential gyrase inhibitors have been identified. According to these screening results, we acquired 235 compounds to analyze their inhibition activities against bacterial DNA gyrase using gel- and SDFQ-based DNA gyrase inhibition assays and discovered 155 new bacterial DNA gyrase inhibitors with a wide structural diversity. Several of them have potent antibacterial activities. These newly discovered gyrase inhibitors include several DNA gyrase poisons that stabilize the gyrase-DNA cleavage complexes and provide new chemical scaffolds for the design and synthesis of bacterial DNA gyrase inhibitors that may be used to combat multidrug-resistant bacterial pathogens. Additionally, this HTS assay can be applied to screen inhibitors against other DNA topoisomerases.

Keywords: DNA gyrase, DNA topoisomerase, gyrase inhibitor, supercoiling-dependent fluorescence quenching assay, high-throughput screening assay, gyrase poison

Antibiotic resistance is a serious threat to modern human society.1−3 It can affect anyone anywhere in the world as the World Health Organization warned (https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance). The United States Centers for Disease Control and Prevention estimated that antibiotic-resistant bacteria infect more than 2.8 million people annually in the United States and, as a result, more than 35,000 Americans die (https://www.cdc.gov/drugresistance/national-estimates.html). Antibiotic resistance is especially high risk to patients who receive cancer chemotherapy or undergo complex surgeries including organ and bone marrow transplantation due to the fact that these patients are much more vulnerable to bacterial infections.1 The emergence of multiple drug-resistant bacteria, so-called “superbugs” that are resistant to almost all antibiotics, makes the situation much worse.2 Without new antibiotics, it is difficult or impossible to treat certain infections by these “superbugs”.3 A pandemic caused by “superbugs” is likely to occur in the future due to the fact that there is no treatment when these diseases emerge.4

Bacterial DNA gyrase, a type II topoisomerase that can introduce (−) supercoils to DNA substrates, is an important and clinically validated target for discovering and developing new antibiotics.5−9 This enzyme has two different subunits, gyrA and gyrB, which form an active A2B2 complex.6 Because DNA gyrase only exists in bacteria and is an essential enzyme to bacteria,10−12 it is possible to identify inhibitors targeting DNA gyrase without affecting human topoisomerases.9,13 Additionally, DNA gyrase can form covalent enzyme-DNA complex intermediates,14−16 a property that makes gyrase an excellent bactericidal target. Indeed, fluoroquinolones (FQs) are among the most successful antibiotics targeted to DNA gyrase.17,18 The mechanism of action (MoA) of FQs is to stabilize the enzyme-DNA cleavage complex, which is ultimately responsible for cell death.19,20 This gyrase poisoning mechanism makes FQs among the most effective and prescribed antibiotics.21,22 Unfortunately, bacterial resistance to FQs has emerged23−27 and makes the development of new, more effective antibiotics an urgent issue, especially for Gram-negative bacterial infections.28 Furthermore, since FQs have been explored extensively, the limit and potential of what FQs can provide likely have been reached.21 Therefore, it is necessary to develop new types of compounds targeting DNA gyrase. Novel bacterial topoisomerase inhibitors (NTBIs) including gepotidacin and zoliflodacin are examples of this effort.29,30 They are bacterial gyrase poisons29 and currently under phase 3 clinical trials for the treatment of gonorrhea.31,32 Nevertheless, more gyrase inhibitors/poisons with novel chemical scaffolds are needed. To achieve this, one may screen chemical compound libraries to yield synthetic entities/scaffolds for further development. One challenge is to develop rapid and efficient high-throughput screening (HTS) assays to identify inhibitors from thousands of compounds in small molecule libraries.

Recently, we pioneered a new method to produce fluorescently labeled, relaxed (Rx) or supercoiled (Sc) DNA molecules to study DNA topoisomerases by supercoiling-dependent fluorescence quenching (SDFQ) or fluorescence resonance energy transfer.33,34 This assay stems from a property of alternating (AT)n sequences in the closed circular plasmids that undergo rapid cruciform formation–deformation depending on the supercoiling status of the plasmids.35,36 The distance between a pair of fluorophore-quencher inserted in the (AT)n sequence is dramatically changed when the plasmids adopt the Sc or Rx form, triggering corresponding changes in the fluorescence intensity.33 These DNA molecules are excellent tools to examine relaxation/supercoiling kinetics of various DNA topoisomerases34 and can be configured into HTS assays to identify topoisomerase inhibitors.33 In this article, we have established an SDFQ-based HTS assay to identify inhibitors targeting bacterial DNA gyrase. After screening the National Institutes of Health’s Molecular Libraries Small Molecule Repository (MLSMR) library containing 370,620 compounds, we identified 155 new bacterial DNA gyrase inhibitors. Several are DNA gyrase poisons, specific DNA gyrase inhibitors that stabilize the gyrase-DNA cleavage-complexes and convert gyrase to a DNA damaging machine.

Results

Establish Miniaturized, Automated SDFQ HTS Assays and Screen the LOPAC Library

Based on the SDFQ assays using the fluorescently labeled Rx plasmid pAB1_FL905 (Figure 1a), we have established a miniaturized, automated primary HTS assay in a 1536-well format to identify bacterial DNA gyrase inhibitors. After a series of experiments, 2 μL of volume, 120 min of incubation time, 175 ng/μL of E. coli DNA gyrase, and 3.21 ng/μL of Rx pAB1_FL905 were optimal and chosen for the assay (Figure 1b,c). The fluorescence intensity was measured at the excitation wavelength of 484 nm and emission wavelength of 520 nm. The assay tolerated up to 4% DMSO without any significant change in signal. We used a known bacterial DNA gyrase inhibitor, novobiocin as the positive control. Results in Figure 1d clearly demonstrate that novobiocin is a potent gyrase inhibitor with an IC50 of 26 nM. Similar conditions were also obtained for a secondary SDFQ HTS assay in which Rx pAB1_FL924 (Figure 1a) was used as the DNA substrate (Figure 1e,f). The excitation wavelength of 531 nm and emission wavelength of 595 nm were used for the secondary HTS assay.

Figure 1.

Figure 1

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SDFQ assays to determine the optimal conditions for the HTS assays. All assays were performed as described for the HTS format in the Materials and Methods section. (a) SDFQ assay catalyzed by DNA gyrase. Plasmid pAB1_FL905 contains fluorescein and dabcyl as the fluorophore and quencher, respectively. Fluorescence was measured using λex = 484 nm and λem = 520 nm. Plasmid pAB1_FL924 carries TAMRA and BHQ2 as the fluorophore and quencher, respectively. Fluorescence was measured using λex = 531 nm and λem = 595 nm. (b) Different amounts of E. coli DNA gyrase were used in the SDFQ assays. (c) Time courses in the presence (red dots) or absence (black dots) of E. coli DNA gyrase (175 ng/mL). 6.42 ng/mL of Rx pAB1_FL905 was used in these assays. (d) Novobiocin potently inhibited E. coli DNA gyrase. The IC50 was determined to be 26 nM as described in the Materials and Methods section. The slope was determined to be 1.462. Assays used 3.21 ng/λL of pAB1_FL905 and 175 ng/λL of E. coli DNA gyrase. Fluorescence was measured using λex = 484 nm and λem = 520 nm. The fluorescence signals of positive and negative controls are 812,515 ± 15,320 and 338,057 ± 26,067, respectively. (e) SDFQ assays with different concentrations of pAB1_FL924 in ng/λL. (f) Novobiocin potently inhibited E. coli DNA gyrase activities. Fluorescence was measured using λex = 531 nm and λem = 595 nm. The x-axis is the logarithm of novobiocin concentrations in molar (M). Different curves represent the incubation time in min.

To validate HTS readiness, we screened the Sigma LOPAC1280 library of 1280 pharmacologically active compounds using the primary HTS assay. Figure 2a and Table 1 show the results. The following are screening statistics: Z′ = 0.70 and 41 hits/compounds of more than 40% inhibition activities with a hit rate of ∼3.2%. The hits include three known gyrase inhibitors (lomefloxacin, ofloxacin, and trovafloxacin) and several known DNA topoisomerase II inhibitors such as suramin,37 aurintricarboxylic acid,38 and emodin.39 Although the Sigma LOPAC1280 library carries two additional DNA gyrase inhibitors, nalidixic acid and oxolinic acid, their IC50 values against E. coli DNA gyrase are more than 30 μM,40 and it is not surprising that they are not included on the hit list since 5 μM of compounds was used. We also noticed that some compounds per se have very strong fluorescence, which results in more than 100% inhibition (Figure 2a and Table S1). They are false positives and should be excluded from the hit list. Unexpectedly, two new E. coli DNA gyrase inhibitors were discovered from the pilot screen: chloro-IB-MECA (Figure 2b) and metergoline (Figure S1a). Their inhibition against E. coli DNA gyrase were confirmed by using agarose gel-based DNA gyrase assays (Figures 2c and S1b). Metergoline is a dopamine agonist and serotonin antagonist41,42 and inhibits gyrase activities at 100 and 200 μM for agarose gel-based assays (Figure S1b). Chloro-IB-MECA is an adenosine analogue and an antagonist of adenosine A3 receptors.43 It potently inhibits E. coli DNA gyrase activities with an IC50 of 2.4 μM (Figure 2c,d). The LOPAC library also contains two similar adenosine analogues: IB-MECA and AB-MECA (Figure S1c). IB-MECA also inhibits E. coli DNA gyrase with an IC50 of 50.7 μM (Figure 2c,d). AB-MECA does not inhibit E. coli gyrase activities at all (Figure 2c). The ATPase assays of E. coli DNA gyrase show that chloro-IB-MECA and IB-MECA are ATP competitive inhibitors of E. coli DNA gyrase (Figure 2e). Interestingly, chloro-IB-MECA did not strongly inhibit E. coli DNA topoisomerase I, E. coli DNA topoisomerase IV, human DNA topoisomerase I, and human DNA topoisomerase IIα (Figure S1d,e). It did not cause gyrase-mediated DNA double-stranded breaks as well (Figure S1f). The experimental results from the pilot screen demonstrate that our SDFQ-based assays are HTS-ready for an HTS campaign to identify new bacterial DNA gyrase inhibitors.

Figure 2.

Figure 2

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(a) The pilot screening of the LOPAC library at 5 μM for E. coli DNA gyrase inhibitors. DMSO and novobiocin were used as negative (yellow triangles) and positive (red squares) controls, respectively. Black dots are the screened compound results. The x-axis represents the catalog numbers of certain compounds from the LOPAC compound library (Sigma-Aldrich). (b) The chemical structure of chloro-IB-MECA (CIBM), a new gyrase inhibitor. (c) Gel-base DNA gyrase assays to confirm that CIBM and IB-MECA (IBM) inhibit E. coli DNA gyrase activities. Lanes 1 and 2 contain relaxed (Rx) and supercoiled (Sc) pAB1. Lanes 3–13 are DNA samples from DNA gyrase assays. Lane 3 contains 200 μM of AB-MECA (ABM). Lanes 4–7 contain 25, 50, 75, and 100 μM of IBM, respectively. Lanes 8–13 contain 0, 5, 10, 20, 50, and 100 μM of CIBM, respectively. (d) SDFQ-based gyrase assays in the presence of CIBM (solid circles) and IBM (open squares). The IC50 values against E. coli DNA gyrase are 2.4 ± 0.9 and 50.7 ± 4.7 μM, respectively. The standard deviations are calculated according to three independent experiments. The x-axis log[CPD] represents the logarithm of compound concentrations in μM. (e) Inhibition of E. coli DNA gyrase ATPase activities by CIBM in the presence of Rx pAB1. Closed circles and open down triangles represent the ATP hydrolysis in the absence and presence of 500 nM of CIBM, respectively. Vmax are 16.3 and 16.6 nM/s for the ATPase activities of gyrase in the absence and presence of 500 nM of CIBM, respectively. KM are 0.59 and 1.35 mM in the absence and presence of 500 nM of CIBM, respectively. The dissociation of the gyrase-CIBM complex Ki is calculated to be 388 nM.

Table 1. SDFQ HTS Screen Statistics.

  pilot screen HTS
no. of compounds 1280 370,620
tested concentration 5 μM 5 μM
Z′ value 0.7 0.81
RZ′ value 0.68 0.83
S/Ba 2.5 2.7
no. of compounds with inhibition >40% 41 2891
hit rate 3.2% 0.78%
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a

S/B represents signal versus background ratio.

Screen the MLSMR Compound Library

We screened the MLSMR compound library containing 370,620 compounds according to the workflow in Figure S2. The SDFQ primary assays were performed using a compound concentration of 5 μM. The screening results are summarized in Figure 3a and Table 1. Specifically, 2891 compounds have more than 40% inhibition activities against E. coli DNA gyrase with a hit rate of 0.78 (Table 1). We retested these 2891 compounds using the primary and secondary assays and found that 929 compounds have more than 32% inhibition activities against E. coli DNA gyrase in both assays with an assumption of up to a 20% variation (Table S2). We also analyzed the fluorescence results and found that some compounds have very high fluorescence at the wavelengths used for the signal detection which gives much more than 100% inhibition activities (Figure 3a and Table S2). These highly fluorescent compounds are false positives and were excluded from the potential DNA gyrase inhibitors.

Figure 3.

Figure 3

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(a) The screening of the MLSMR compound library for E. coli DNA gyrase inhibitors. DMSO and novobiocin were used as negative and positive controls, respectively. The data are trimmed with 531 compounds >250% activities not shown. (b) 155 new gyrase inhibitors have an IC50 < 200 μM against E. coli DNA gyrase, and 33 compounds have an IC50 < 15 μM.

We decided to conduct a more detailed analysis for the top 218 hits with gyrase inhibition activities between 50 and 120% in both primary and secondary assays (Table S3). Among these 218 compounds include 25 known gyrase inhibitors, such as novobiocin and ciprofloxacin, and 82 DNA intercalators or potential DNA intercalators, such as 9-aminoacridine,44 echinomycin,45 and several anthracyclines.46 When DNA intercalators bind to (−) Sc plasmid pAB1_FL905, they significantly unwind the plasmid DNA molecule and temporarily convert it into the relaxed or (+) supercoiled DNA molecule.47 As a result, the AT hairpin structure is converted into the double-stranded form,36 and the fluorescence intensity of pAB1_FL905 is greatly enhanced. Figure S3 shows two examples in which ethacridine and echinomycin, DNA intercalators, significantly increase the fluorescence intensity of (−) Sc pAB1_FL905 upon binding to (−) Sc pAB1_FL905. Please note that ethacridine and echinomycin does not have fluorescence per se under this experimental condition. Interestingly, both intercalators also inhibited E. coli DNA gyrase activities at higher concentrations (Figure S3c,d), likely through DNA binding and blocking gyrase accessing to the plasmid DNA molecules. Due to this reason, we consider DNA intercalators as “false” positives. Detecting 25 known gyrase inhibitors among the 218 top hits demonstrates the success of the SDFQ HTS campaign.

Novel DNA Gyrase Inhibitors

We purchased/obtained 235 compounds (Table S4) for further analyses based on the screening results and following selection criteria: (1) Compounds have similar inhibition activities against E. coli DNA gyrase in both primary and secondary assays; (2) compounds with more than 150% inhibition activities against DNA gyrase are excluded; (3) known DNA gyrase and topoisomerase II inhibitors are also excluded; (4) DNA intercalators or potential DNA intercalators are generally not selected; and (5) we did not specifically exclude pan-assay interference compounds (PAINS). After we confirmed the identities of these compounds using mass spectrometry, agarose gel-based assays were used to determine their inhibition activities against E. coli DNA gyrase (Figure S4). Our results showed that 155 compounds are E. coli DNA gyrase inhibitors with an IC50 value of <200 μM (Figure S4). Among these 155 new gyrase inhibitors, 77 have an IC50 of <50 μM (Figure S4). Agarose gel-based and SDFQ-based titration experiments were used to determine the IC50 values of these 77 new DNA gyrase inhibitors and showed that 45 have an IC50 of <25 μM and 33 have an IC50 of <15 μM (Figure 3b, Figure S5, and Table S4).

According to their chemical structures, these 155 new gyrase inhibitors can be divided into 10 groups: (1) psoralen derivatives, (2) quinazoline derivatives, (3) dihydroxynaphthalene-2-carboxylate and quinolinedione derivatives, (4) isatin-phenylhydrazone derivatives, (5) amino-benzothiazole derivatives, (6) thiazolo[3,2-a]benzimidazole derivatives, (7) pyrido-thieno-pyrimidine derivatives, (8) compounds containing a rhodamine moiety, (9) fluorone derivatives, and (10) other compounds. We conducted a more detailed study for several new gyrase inhibitors and determined their MoA. For instance, compound 154, N-(6-chloro-4-phenylquinazolin-2-yl)guanidine, is a quinazoline derivative (Figure 4a) and strongly inhibits E. coli DNA gyrase activities with an IC50 of approximately 7 μM (Figure 4b,c). Intriguingly, compound 154 also causes the gyrase-mediated DNA double-stranded breaks and single-stranded nicks (Figure 4d). A likely MoA of this gyrase inhibitor is to stabilize the enzyme-DNA cleavage-complex, which leads to the DNA breaks and nicks. In other words, compound 154 is a bacterial DNA gyrase poison. Although the induced DNA breaks and nicks are generally proportional to the added inhibitor, high concentrations of compound 154 inhibit the formation of the double-stranded DNA breaks (compare lanes 4 to 6 of Figure 4d). Since human DNA topoisomerase IIα is a type IIA topoisomerase,7,48 we also determined whether compound 154 inhibits human DNA topoisomerase IIα and found that it inhibits human DNA topoisomerase IIα with an estimated IC50 of ∼50 μM (Figure 4e). As expected, compound 154 also causes the human topoisomerase IIα-mediated DNA nicks and double-stranded breaks (Figure 4f). Apparently, compound 154 is a human DNA topoisomerase IIα poison as well.

Figure 4.

Figure 4

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Compound 154 is a bacterial DNA gyrase poison. (a) Chemical structure of compound 154. (b) SDFQ-based gyrase assays in the presence of compound 154 (open squares) and novobiocin (stars). The IC50 values against E. coli DNA gyrase are 3.1 ± 0.7 μM. The standard deviations are calculated according to three independent experiments. (c) Agarose gel-based gyrase inhibition assays for compound 154. Lanes 3–8 correspond to 1.56, 3.12, 6.25, 12.5, 25, and 50 μM of the compound, respectively. Lanes 1 and 2 are relaxed and supercoiled plasmid pAB1, respectively. (d) Gyrase-mediated DNA cleavage assays were performed as described in the Materials and Methods section using plasmid pBR322. Lane 1 does not contain a gyrase inhibitor. Lanes 2–5 contain 5, 25, 50, 100, and 200 μM of compound 154, respectively. Lane 7 contains 50 μM of ciprofloxacin (CFX). (e) Agarose gel-based inhibition assays against human DNA topoisomerase IIα for compound 154. Lanes 3–6 correspond to 0, 25, 50, and 100 μM of the compound, respectively. Lanes 1 and 2 are supercoiled and relaxed plasmid pAB1, respectively. (f) Human DNA topoisomerase IIα-mediated DNA cleavage assays were performed as described in the Materials and Methods section using plasmid pBR322. Lanes 1–3 contain 50, 100, and 200 μM of compound 154, respectively. Lane 4 contains 100 μM of etoposide (ETP). Lanes 5 and 6 contain DNA samples from the assay mixtures in the absence of ETP and human DNA topoisomerase IIα, respectively. Symbols Rx, Sc, Nk, and Ln represent relaxed, supercoiled, nicked, and linear DNA, respectively.

Compound 40, 1-[(4-carbamoylphenyl)carbamoyl]ethyl 1,4-dihydroxynaphthalene-2-carboxylate (Figure 5a), also inhibits E. coli DNA gyrase activities with an IC50 of 50 μM (Figure 5b,c). Similar to compound 154, it causes gyrase-mediated DNA double-stranded breaks and single-stranded nicks and is a bacterial DNA gyrase poison (Figure 5d). We noticed that compound 40 causes many more DNA nicks than double-stranded DNA breaks (compare lanes 1 to 4 of Figure 5d). Compound 40 does not inhibit human DNA topoisomerase IIα (Figure 5e). It does not cause the human topoisomerase IIα-mediated DNA nicks and double-stranded breaks (Figure 5f).

Figure 5.

Figure 5

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Compound 40 is a bacterial DNA gyrase poison. (a) Chemical structure of compound 40. (b) SDFQ-based gyrase assays in the presence of compound 40 (open circles) and novobiocin (solid stars). The IC50 values against E. coli DNA gyrase are 47.6 ± 3.7 μM. The standard deviations are calculated according to three independent experiments. (c) Agarose gel-based gyrase inhibition assays for compound 40. Lanes 3–9 correspond to 6.25, 12.5, 25, 50, 100, 150, and 200 μM of compound 40, respectively. Lanes 1 and 2 are relaxed and supercoiled plasmid pAB1, respectively. (d) Gyrase-mediated DNA cleavage assays were performed as described in the Materials and Methods section using plasmid pBR322. Lanes 6 and 1–4 contain 0, 25, 50, 100, and 200 μM of compound 40, respectively. Lane 5 contains 50 μM of ciprofloxacin (CFX). (e) Agarose gel-based inhibition assays against human DNA topoisomerase IIα for compound 40. Lanes 3–6 correspond to 12.5, 25, 50, and 100 μM of compound 40, respectively. Lanes 1 and 2 are relaxed and supercoiled plasmid pAB1, respectively. (f) Human DNA topoisomerase IIα-mediated DNA cleavage assays were performed as described in the Materials and Methods section using plasmid pBR322. Lanes 1 and 2 contain 100 and 200 μM of compound 40, respectively. Lane 3 contains 100 μM of etoposide (ETP). Lanes 4 and 5 contain DNA samples from the assay mixtures in the absence of etoposide and human DNA topoisomerase IIα, respectively. Symbols Rx, Sc, Nk, and Ln represent relaxed, supercoiled, nicked, and linear DNA, respectively.

Among these new DNA gyrase inhibitors are 12 psoralen derivatives. A preliminary structure–activity relationship has been determined. Figures 6 and S6 show our results. All psoralen derivatives except compounds 118 and 122 inhibited E. coli DNA gyrase activities. The 9-methyl group enhances the anti-DNA gyrase potency. A bulky hydrophobic group at the third position and a carboxyl group at the sixth position are required for the anti-DNA gyrase activities. Our ATPase assays showed that all psoralen derivatives are ATP competitive inhibitors of DNA gyrase (Figure S7). A further analysis of compound 48 shows that it inhibited the ATPase activities of E. coli DNA gyrase with a Ki (the dissociation constant for the inhibitor) value of 150 nM (Figure S7). These psoralen derivatives did not greatly inhibit E. coli DNA topoisomerase I and human DNA topoisomerase IIα with IC50 > 100 μM (Figure S7). They did not cause gyrase-mediated DNA cleavage and are not DNA poisons (data not shown).

Figure 6.

Figure 6

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Psoralen derivatives are potent gyrase inhibitors with antibacterial activities. The IC50 against E. coli were determined using the SDFQ- and confirmed with gel-based gyrase inhibition assays.

Antibacterial Activities

We carried out antibacterial studies for compounds 40 and 154 and the 12 psoralen derivatives. Both Gram-positive and -negative strains are included. Table 2 shows our results. Compound 154 showed significant antibacterial activities against all bacterial strains including the wild-type Escherichia coli strain ATCC 25922, Staphylococcus aureus ATCC 14775, and methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591. Compound 40 showed anti-Bacillus subtilis activities with an minimal inhibitory concentration (MIC) value of 100 μM. Certain psoralen derivatives showed potent antibacterial activities against Staphylococcus aureus ATCC 14775 and MRSA ATCC 33591. For instance, the MIC values of compounds 119 and 120 against Staphylococcus aureus ATCC 14775 and MRSA ATCC 33591 are 0.39 and 0.78 μM, respectively. Intriguingly, the antibacterial activities of these psoralen derivatives are correlated with the anti-DNA gyrase activities. As shown above, the 9 methyl group enhances the antigyrase potency and is also required for the antibacterial activities against S. aureus and MRSA. Likewise, a bulky hydrophobic group at the third position and a carboxyl group at the sixth position are required for the antigyrase and antibacterial activities as well. Although compound 125 potently inhibited E. coli DNA gyrase, it did not inhibit the growth of S. aureus and MRSA (Table 2). Possibly, the amine in the bulky hydrophobic group at the third position prevented the entry of the compound to bacterial cells. Further studies are needed to confirm this hypothesis.

Table 2. Minimal Inhibitory Concentrations (MIC) against Various Bacterial Strains by Microbroth Dilution.

  MIC (μM)
compd no. E.coli ATTC 25922 E. coli imp BAS3023 S. aureus ATCC 14755 MRSA ATCC 33591 B. subtilis ATCC 6633
40 >200 >200 >200 >200 100
154 50 25 12.5–25 12.5–25 50
25 >200 >200 >200 >200 25
46 >200 >200 >200 >200 6.25
48 >200 >200 3.125 3.125 >200
117 >200 >200 0.78 0.78 >200
118 >200 >200 200 200 >200
119 >200 >200 0.39 0.78 >200
120 >200 >200 0.39 0.78 >200
121 >200 >100 3.125 3.125 50
122 >200 >200 200 200 >200
123 >200 >200 1.56 1.56 6.25
124 >200 >200 1.56 1.56 25–50
125 >200 >200 >200 >200 >200
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Discussion

We have established a miniaturized, automated HTS assay based on the SDFQ assay33,34 to identify E. coli DNA gyrase inhibitors. This HTS assay demonstrated excellent statistics for two screens: a pilot screen of the LOPAC library and a full HTS campaign of the MLSMR library with Z′ values at 0.7 and 0.81, respectively (Table 1). After the pilot screen, we discovered two new gyrase inhibitors: chloro-IB-MECA (Figure 2b) and metergoline (Figure S1a). Since chloro-IB-MECA is an adenosine analogue and a potent gyrase inhibitor (Figure 2c,d), this result suggests that adenosine analogues have potential to be developed into antibiotics targeting bacterial DNA gyrase. More importantly, after the HTS campaign of screening the MLSMR compound library, we discovered 155 new gyrase inhibitors that provide novel and diverse chemical structures and scaffolds for further development.

This new HTS assay has several advantages. First, it can be automated for HTS studies, an advantage over existing screening assays.49−52 Our successful HTS campaign of screening the MLSMR library attests the power and potential of this assay. This HTS assay not only can be used to screen and identify bacterial DNA gyrase inhibitors but also can be applied to screen and identify inhibitors of other DNA topoisomerases, such as bacterial DNA topoisomerase I, human DNA topoisomerase 1, and human DNA topoisomerase IIα. Second, unlike previous HTS efforts mainly targeted the gyrB ATPase domain of bacterial gyrase,53 this HTS assay, employed relaxed DNA as the substrate, would identify inhibitors that affect all possible steps of the catalytic cycle of E. coli DNA gyrase, including DNA binding, DNA cleavage, strand passage, and DNA relegation. For example, ethacridine and echinomycin are two DNA intercalators and bind to DNA with high affinity.45,54 They also potently inhibited E. coli DNA gyrase activities (Figure S3c,d), although DNA intercalation/unwinding of pAB1_FL905 by these two intercalators interferes with the fluorescence signal detection (Figure S3a,b). In fact, many DNA intercalators are among the hits of this HTS campaign (Table S3). It is likely that DNA binding by DNA intercalators is the inhibition MoA. As discussed above, we identified more than 10 different groups of E. coli DNA gyrase inhibitors. While chloro-IB-MECA and psoralen derivatives are competitive inhibitors of E. coli DNA gyrase (Figures 2e and S7), compounds 40 and 154 stabilize the gyrase-DNA cleavage complex and cause DNA double-stranded breaks and single-stranded nicks (Figures 4 and 5). Additionally, this HTS assay is a cost-effective assay and only uses 6.42 ng in 2 μL per assay/well in 1536 well plates. Approximately 2.5 mg of Rx pAB1_FL905 was used to screen 370,620 compounds in the MLSMR library for the primary assay. It is feasible to conduct more screens in the future for bacterial DNA gyrase and other DNA topoisomerases.

A major difficulty of this HTS assay is the fluorescence interference caused by certain compounds that have very strong fluorescence per se. This interference results in over 100% inhibition. We are considering these fluorescent compounds as false positives. However, some compounds with high fluorescence may be gyrase inhibitors and will be missed here. An orthogonal screening assay using a different mechanism, such as the T5 exonuclease-based HTS assays,55 may be used to identify these gyrase inhibitors. Nevertheless, as stated above, the HTS assay is an excellent assay to identify inhibitors targeting the catalytic cycle of DNA topoisomerases including DNA gyrase. It cannot differentiate catalytic inhibitors and poisons, although DNA topoisomerase poisons also inhibit the catalytic activities of DNA topoisomerases. Gel-based DNA cleavage assays are required to identify DNA topoisomerase poisons. Indeed, 8 DNA gyrase poisons have been identified in this study (Table S4).

The most exciting result of this HTS campaign is the discovery of 155 new DNA gyrase inhibitors (Table S4). Some of them showed potent antibacterial activities (Table 2), and eight are gyrase poisons (Table S4). For instance, compound 154, a quinazoline derivative, is a new DNA gyrase poison with significant antibacterial activities (Figure 4 and Table 2). Structurally, this new gyrase poison is different from quinolones17 and quinolone-like gyrase poisons, quinazolinediones.56,57 It does not carry a quinone moiety and a carboxy group that can interact with the quinolone-resistance-determining region of DNA gyrase,58,59 suggesting that compound 154 may overcome quinolone-resistant mutations.59 In other words, compound 154 provides a promising new scaffold for the development of novel antibiotics to treat FQ-resistant bacterial infections.59 Compound 154 is a small-size molecule with a molecular weight of 297.75 Da and should have great potential for derivatization and bacterial cell penetration.53 According to its chemical structure, it is likely that compound 154 binds to bacterial DNA gyrase through gyrase-mediated DNA intercalation.59 Indeed, our molecular modeling results show that compound 154 nicely intercalates into DNA base pairs near the gyrase cleavage sites in the gyrase-DNA-drug complex although compound 154 does not directly interact with DNA gyrase (Figure 7a,b). Interestingly, compound 154 is also a human DNA topoisomerase IIα poison with a higher IC50 value. This is not very surprising since bacterial DNA gyrase and human DNA topoisomerase IIα belong to type 2A topoisomerases.7,48 Previous results showed that certain FQs and quinazoline derivatives are human DNA topoisomerase IIα poisons too.60,61 The scaffold of compound 154 may be developed into specific bacterial gyrase or human topoisomerase IIα inhibitors/poisons depending on design and derivatization.

Figure 7.

Figure 7

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Molecular models of compounds 154 (a and b) and 40 (c and d) binding to gyrase-DNA complexes. Molecular models were constructed as described in the Materials and Methods section. (a) Compound 154 is shown in space fill model. (b) Compound 154 (stick model) intercalates between DNA base pairs (space fill models). (c) Compound 40 is shown in space fill model. (d) Compound 40 (stick model) intercalates between DNA base pairs (green).

Compound 40, a 1,4-dihydroxy-2-naphthoic acid derivative, is another new E. coli DNA gyrase poison discovered in this study (Figure 5). Although it does not strongly inhibit E. coli DNA gyrase activities (Figure 5b,c), compound 40 induces the formation of gyrase-mediated DNA nicks and double-stranded breaks (Figure 5d). Noticeably, it induces much more single-stranded DNA nicks than the double-stranded breaks (Figure 5d). Compound 40 carries a fused six-member aromatic ring system with a delocalized conjugated π system that may intercalate into DNA base pairs, connecting to a benzamide group through a flexible linear linker (Figure 5a). This structure feature is similar to that of NBTIs,30 although the linker is shorter. We therefore performed a molecular modeling studies based on a cryoEM structure of E. coli DNA gyrase nucleoprotein complex with gepotidacin, an NTBI.62 Our molecular modeling results show that the fused six-member aromatic ring system intercalates into DNA base pairs of the nicking site (Figure 7c,d), and the benzamide group lies on the floor of the major groove (Figure 7c,d), unlike the binding of gepotidacin to the gyrase nucleoprotein complex likely due to the short linker of compound 40. In contrast to compound 154, compound 40 does not inhibit human DNA topoisomerase IIα and cause topoisomerase IIα-mediated DNA nicks and double-stranded breaks as well (Figure 5c,d). In other words, compound 40 is a specific gyrase poison. Among the 155 new gyrase inhibitors, compounds 81, 205, and 234 have similar chemical structures and also cause gyrase-mediated DNA nicking (Table S4).

Many of these newly discovered gyrase inhibitors are catalytic inhibitors of E. coli DNA gyrase including chloro-IB-MECA and psoralen derivatives. Chloro-IB-MECA is the first adenosine analogue gyrase inhibitor discovered so far. As expected, chloro-IB-MECA is an ATP competitive inhibitor of E. coli DNA gyrase. Nevertheless, chloro-IB-MECA does not strongly inhibit other DNA topoisomerases. Some minor modifications significantly affect the IC50 values against E. coli DNA gyrase, suggesting specific interactions exist between chloro-IB-MECA and E. coli DNA gyrase. These results suggest that adenosine analogues should be explored as specific gyrase inhibitors in the future although previous campaigns to identify antibiotics targeting the ATPase domain of bacterial DNA gyrase have not been successful.53 Psoralen derivatives are another type of ATP competitive inhibitor of E. coli DNA gyrase. Similar to chloro-IB-MECA, certain modifications are critical for their antigyrase activities. Figure S8 shows our molecular modeling results of these two new gyrase inhibitors binding to the ATP binding pocket of gyrB. Both compounds form a hydrogen bond with residue Asp73 of gyrB.53,63

MIC results showed that compound 154 has potent antibacterial activities against E. coli, S. aureus, MRSA, and B. subtilis (Table 2) and has potential to be developed into broad-spectrum antibiotics. Our preliminary MTT cell viability results, however, show that compound 154 is cytotoxic toward human A549 cells (data not shown). Future medicinal chemistry modifications should focus on synthesizing and selecting derivatives with less cytotoxicity. Since compound 154 is also a human DNA topoisomerase IIα poison (Figure 4f), this compound and derivatives can be developed into anticancer drugs. Further studies are required. We also noticed that compound 40 and 12 psoralen derivatives do not have strong antibacterial activities against Escherichia coli strains ATCC 25922 and BAS3023 (Table 2). It is possible that these compounds cannot cross the E. coli cell envelope and inhibit DNA gyrase in vivo. Additionally, the lack of antibacterial activity of compound 40 may stem from the fact that it is an ester and might be hydrolyzed in cell-based assays. More studies are needed to confirm this possibility. Nevertheless, certain psoralen derivatives have potent antibacterial activities against S. aureus, MRSA, and B. subtilis (Table 2). More importantly, their antibacterial activities correlate with their anti-DNA gyrase activities. As stated above, a bulky hydrophobic group at the third position and a carboxyl group at the sixth position are required for their antigyrase activities. Interestingly, these two groups are also required for their antibacterial activities, indicating that the DNA gyrase is the cellular target of these psoralen derivatives.

In summary, we have developed an HTS assay based on the SDFQ assay and screened the LOPAC and MLSMR libraries. After the screen, we identified 155 new bacterial DNA gyrase inhibitors including catalytic inhibitors and gyrase poisons. Not only can this HTS assay be applied to screen inhibitors against other DNA topoisomerases, but also the new gyrase inhibitors provide a wealth of chemical structures/scaffolds to develop new generations of antibiotics targeting bacterial DNA gyrase that combat multidrug-resistant bacterial pathogens.

Materials and Methods

Proteins and Plasmids

E. coli DNA gyrase was purified according to the purification procedures published previously.64 His-tagged human DNA topoisomerase IIα C-terminal deletion mutant (hTopo2α-ΔCTD) that lacks the 310 aa residues in the C-terminus was purified using a Ni-NTA column from yeast strain RegΔ1 carrying plasmid YEpWobb-hTopo2α-ΔCTD. Plasmid YEpWobb-hTopo2α-ΔCTD is a gift from Dr. Nei-Li Chan at National Taiwan University. E. coli DNA topoisomerase I was purified as described previously.65E. coli DNA topoisomerase IV is a gift from Keir Neuman at NIDDK. Pyruvate kinase/lactic dehydrogenase enzymes from rabbit muscle were purchased from Sigma-Aldrich, Inc. Plasmid pAB1 was described previously.33 Fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 were synthesized as described previously.33

Chemicals

LOPAC compound library, phosphoenol pyruvate, and NADH were purchased from Sigma-Aldrich, Inc. The MLSMR compound library was a compound collection at the Sanford Burnham Prebys Medical Discovery Institute. Vendors and sources of other chemical compounds are shown in Table S4. The identities of these chemical compounds were confirmed using mass spectrometry.

SDFQ HTS Assay

Using pAB1_FL905, the assay was performed in 2 μL of 1 × DNA gyrase buffer (20 mM Tris-Acetate pH 7.9, 50 mM KAc, 10 mM MgCl2, 2 mM DTT, 1 mM ATP, 0.1 mg/mL BSA). The following is the procedure: (1) Using the BioRAPTR, dispensed 1 μL of E. coli DNA gyrase (350 ng/ μL) with a final concentration in assay 175 ng/μL. (2) Using the BioRAPTR, dispensed 1 μL of DNA Rx pAB1_FL905 (6.425 ng/μL); final concentration in assay is 3.2125 ng/μL in assay. (3) Spun plate at 800 rpm for 30 s. (4) Incubated the plate at 37 °C for 2 h in the dark and read the plate on the Envision measuring fluorescence (excitation@484 nm, emission@Em520). Z′-factor was calculated using the following equation:

graphic file with name pt2c00113_m001.jpg

where σp, σn, μp, and μn represent the sample standard deviations and means for positive (p) and negative (n) controls, respectively. The robust Z′-factor (RZ’) was calculated using the medians rather than the means.

SDFQ HTS Assay

Using pAB1_FL924, the assay was performed in 2 μL of 1 × DNA gyrase buffer. The following is the procedure: (1) Using the BioRAPTR, dispensed 1 μL of E. coli DNA gyrase (350 ng/μL) with a final concentration in assay 175 ng/μL. (2) Using the BioRAPTR, dispensed 1 μL of DNA Rx pAB1_FL924 (6.425 ng/μL); final concentration in assay is 3.2125 ng/μL in assay. (3) Spun plate at 800 rpm for 30 s. (4) Incubated the plate at 37 °C for 2 h in the dark and read the plate on the Envision measuring fluorescence (excitation at 531 nm, emission at Em595).

SDFQ-based DNA Gyrase Inhibition Assays

The assays were performed in 30 μL of 1 × gyrase buffer containing 400 ng of of Rx pAB1_FL905 at 37 °C. 100 ng of DNA gyrase (8.9 nM) was used to supercoil the Rx pAB1_FL905 in the presence of different concentrations of a gyrase inhibitor. The fluorescence intensity at λem = 521 nm was monitored with λex = 494 nm in a microplate reader. The IC50 values were estimated by nonlinear fitting of the following equation: Inline graphic, where F is the fluorescence intensity at the x concentration of an inhibitor, Fmax and Fmin are the maximum and minimum fluorescence of the DNA sample, respectively, and P is a slope parameter. Novobiocin was used as a positive control.

Agarose Gel-Based DNA Gyrase Inhibition Assays

The assays were performed in 30 μL of 1 × gyrase buffer containing 400 ng of of Rx pAB1 at 37 °C. 100 ng of DNA gyrase was used to supercoil the Rx pAB1 in the presence of different concentrations of a gyrase inhibitor. After 15 min of incubation with the inhibitor at 37 °C, all reactions were stopped with 1 μL of stop solution (3% SDS and 250 mM EDTA). Samples were analyzed by electrophoresis in 1% w/v agarose gels followed by ethidium bromide staining and photographed under UV light. Novobiocin was used as a positive control.

DNA Gyrase-Mediated DNA Cleavage Assay

250 ng of plasmid pBR322 and 20 nM of E. coli DNA gyrase were mixed and incubated in 1 × gyrase buffer at 37 °C for 15 min in the presence of an inhibitor or compound. After the incubation, 0.2% SDS and 0.1 mg/mL proteinase K were added to the reaction mixtures to trap the gyrase-inhibitor-DNA complex and digest the gyrase, respectively, by incubating for an additional 30 min at 37 °C. DNA samples were analyzed in 1% agarose gel containing 0.5 μg/mL ethidium bromide in 1 × TAE buffer and photographed under UV light. Ciprofloxacin was used as a positive control.

Minimum Inhibitory Concentrations Assays

Antibacterial MICs were obtained from three independent experiments using broth microdilution methods in 96-well plates according to Clinical and Laboratory Standards Institute guidelines. Cells were grown from a singles colony in MHIIB medium for 18 h at 37 °C in agitation. The cultures were then diluted to OD600 of 0.1 using MHIIB medium. 50 μL of the diluted cultures were added to 50 μL of serially diluted compounds in MHIIB in a 96-well plate and incubated at 37 °C for another 18–20 h. After OD600 was measured to monitor the inhibition, and 0.02% resazurin was added to each well and incubated for 4 h at 37 °C. After the incubation, fluorescence with a wavelength of 494 and 521 nm for excitation and emission, respectively, was measured using a plate reader and used to calculate the MIC values. Bacterial strains S. aureus (ATCC 14775), MRSA (ATCC BAA44), B. subtilis (ATCC 6633), E. coli (ATCC 25922), and E. coli BAS3023 that carries an imp4213 mutation66 were used to determine the MICs. Ciprofloxacin and novobiocin were used as positive controls.

E. coli DNA Gyrase ATPase Assays

The assays were performed in 60 μL of 1 × gyrase ATPase buffer containing 50 nM of E. coli DNA gyrase or gyrB, 200 ng of Rx pAB1, 0.8 mM of Phosphoenol pyruvate, 1.2 units of pyruvate kinase, 1.7 units of lactate dehydrogenase, and 0.4 mM of NADH at 37 °C. After the reaction mixtures were incubated 37 °C for 5 min, 2 mM of ATP was added to initiate the reaction. Absorbance at 340 nm was used to monitor the ATPase activities at 37 °C in a spectrophotometer.

Molecular Dynamics Simulation

In order to get an accurate binding model of all selected compounds with E. coli DNA gyrase, we first performed molecular dynamics (MD) simulations to generate multiple conformations of the E. coli DNA gyrase subunit A and subunit B, respectively. Considering the different possible binding models of drugs on gyrA subunit, we constructed two gyrA subunits with short DNA segments that were nicked on both strands but at different positions to mimic the DNA double-strand break from FQ binding and NBTIs binding. Using the Charmm-Gui web interface,67 the three subunits were solvated in a cubic water box with TIP3 water, and the system was neutralized by adding 0.15 M of MgCl2. The final systems contained ∼31,000 (gyrB), ∼218,000 (gyrA-FQ), and ∼222,000 (gyrA-NBTI) atoms respectively. NAMD 2.1468 was used to perform all-atom molecular dynamics with CHARMM36m69 force field. The particle mesh Ewald method70 was used for calculating the long-range ionic interactions. The systems were minimized for 100,000 steps, followed by a 250 ps equilibration at 310 K with 1 fs time step. 200 ns (gyrB) and 100 ns (gyrA) production simulation with 2 fs time step were performed at a constant pressure of 1 atm and T = 310 K. The Nose–Hoover Langevin piston method71 was used for pressure coupling, with a piston period of 50 fs and a decay of 25 fs, and the Langevin temperature coupling with a friction coefficient of 1 ps–1 was used for maintaining the temperature. From the simulation trajectory, 1000 protein pdb frames for gyrB and 500 protein pdb frames for both gyrA systems were extracted using Visual Molecular Dynamics72 for further in silico docking studies. After docking, another 100 ns MD simulation was performed for each protein–drug complex with the same input settings as described above.

In Silico Docking Studies

Chloro-IB-MECA and compound 48 were docked to 1000 MD-generated gyrB conformations. Compounds 40 and 154 were, respectively, docked to 500 MD-generated gyrA conformations (NBTI nicking model) and 500 MD-generated gyrA conformations (FQ nicking model). The protein pdb files and compounds structure were first converted to pdbqt format for docking. AutoDock Vina 1.1.273 was used to perform molecular docking. Using custom scripts, compounds were screened against the protein conformations, and the resulting scores of the complexes were sorted and ranked according to their binding affinities.

Acknowledgments

We would like to thank Dr. Nei-Li Chan for providing plasmid YEpWobb-hTopo2α-ΔCTD and Dr. Keir Neuman for providing E. coli DNA topoisomerase IV. We also thank Dr. Yuk-Ching Tse-Dinh for critical reading of the manuscript and Dr. Thirunavukkarasu Annamalai for help with the MIC assays. We would like to thank the NCI Development Therapeutics Program (DTP) for providing certain compounds. This work was supported by National Institutes of Health grant 1R21AI125973 (to F.L.). L.H.S., S.V. and B.L.H. acknowledge support from the Florida Translational Research Program (FL DOH #COHK8)

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00113.

  • New gyrase inhibitors chloro-IB-MECA, IB-MECA, AB-MECA, and metergoline identified in the pilot screen; the workflow of the HTS assay screening the MLSMR library; DNA intercalation causes fluorescence changes to plasmid pAB1_FL905; agarose gel-based inhibition assays against E. coli DNA gyrase; SDFQ- and agarose-gel based titration assays to determine gyrase IC50; E. coli gyrase inhibition assays with psoralen derivatives; inhibition of ATPase activities of E. coli DNA gyrase by psoralen derivatives; molecular modeling analysis of Cl-IB-MECA and compound 48 binding to the ATP binding pocket of E. coli DNA gyrase B (PDF)

  • Tables S1–S4. Table S1:E. coli DNA gyrase inhibitors identified by screening the LOPAC library. Table S2: E. coli DNA gyrase inhibitors identified by screening the MLSMR library. Table S3: E. coli DNA gyrase inhibitors identified by screening the NCATS library with 50–120% inhibition activities. Table S4: 235 compounds purchased/obtained for analyses (XLSX)

Author Present Address

∥ Alchem Laboratories Corporation, 13305 Rachael Blvd, Alachua, Florida 32615, United States

Author Contributions

F.L. designed the research. E.E.A.M., Z.D., D.A.B., B.L.H., P.C., and F.L. performed the research. F.L., S.V., L.H.S., J.W.C., and P.C. analyzed the data. F.L. wrote the paper.

The authors declare the following competing financial interest(s): A provisional patent application has been filed for the newly identified gyrase inhibitors. L.H.S. is an employee of Fate Therapeutics. S.V. and B.S.H. are employees of Alchem Laboratories.

Supplementary Material

pt2c00113_si_001.pdf (12.8MB, pdf)
pt2c00113_si_002.xlsx (1.8MB, xlsx)

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