Enhancing the Potency of Nalidixic Acid toward a Bacterial DNA

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Enhancing the Potency of Nalidixic Acid Towards a Bacterial DNA Gyrase with Conjugated Peptides Marya Ahmed, and Shana O. Kelley ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00540 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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ACS Chemical Biology

Enhancing the Potency of Nalidixic Acid Towards a Bacterial DNA Gyrase with Conjugated Peptides Marya Ahmed and Shana O Kelley* Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, and Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada *corresponding Author. email: [email protected]

ABSTRACT Quinolones and fluoroquinolones are widely used antibacterial agents. Nalidixic acid (NA) is a first-generation quinolone-based antibiotic that has a narrow spectrum and poor pharmacokinetics. Here, we describe a family of peptideNalidixic acid conjugates featuring different levels of hydrophobicity and molecular charge prepared by solid-phase peptide synthesis that exhibit intriguing improvements in potency. In comparison to NA, which has a low level of potency in S. aureus, the NA peptide conjugates with optimized hydrophobicities and molecular charges exhibited significantly improved antibacterial activity. The most potent NA conjugate - featuring a peptide containing cyclohexylalanine and arginine - exhibited efficient bacterial uptake and notably, specific inhibition of S. aureus DNA gyrase. A systematic study of peptide-NA conjugates revealed that a fine balance of cationic charge and hydrophobicity in an appendage anchored to the core of drug is required to overcome the intrinsic resistance of S. aureus DNA gyrase towards this quinolone-based drug.

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The marked increase in drug-resistant microbes has led to warnings from the World Health Organization (WHO) of a post-antibiotic era, when microbes will be completely resistant to treatment with available antibiotics.1 This alarming prediction is based on the fact that the rate of antimicrobial resistance is overtaking the rate at which new drugs or targets are being discovered.2 Thus, the development of new drugs, and the discovery of new antibacterial targets is the focus of intense effort.2,3 Methicillin-resistant Staphylococcus aureus (MRSA) alone is responsible for 18,000 deaths per year in the United States.4 In addition to the discovery of new antimicrobial targets, the rapidly growing problem of antibacterial resistance must be addressed.2,5-7 The development of novel bacterial topoisomerase inhibitors (NBTIs) is of significant interest to provide new antibiotic candidates.5-7 Quinolones are traditional NBTIs, but these drugs show limited activity towards S. aureus due to the presence of drug-resistant domains in the type II topoisomerases that include DNA gyrase and topoisomerase IV.8 Fluoroquinolones are modified quinolones that are abundantly used due to their broad spectrum and high antibacterial efficacies.8 Emerging resistance to fluoroquinolones in both gram-positive and gram-negative bacteria began in 2001, and is rising yearly.3 Prokaryotic type II topoisomerases are enzymes that are involved in cellular processes including transcription, replication, chromosome condensation and segregation.5,9 DNA gyrase is a tetramer and this enzyme alters the topological state of DNA by creating transient double-stranded breaks in a DNA duplex, followed by the passage of another DNA duplex through the break and then religation of the cleaved DNA duplex. DNA gyrase catalyzes negative supercoiling of closed circular DNA, whereas topoisomerase IV relaxes positive and negative supercoils of DNA.3 Quinolones and fluoroquinolones inhibit the catalytic activity of these enzymes and stabilize the transient DNA breaks formed by bacterial topoisomerases.3,10 The accumulation of transient DNA breaks in bacterial chromosomes prevents bacterial growth by inhibition of DNA transcription, translation and the induction of the SOS response, leading to bacterial death.9-11 However, gram-positive bacteria like S. aureus are inherently


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resistant to first-generation quinolones, due to the overexpression of quinolonespecific drug efflux pumps,11 and inherent mutations in quinolone resistance determining regions (QRDR).9,11,12 Nalidixic acid (NA) is a first-generation quinolone NBTI, which has limited clinical utility due to its restricted antimicrobial spectrum, ease with which bacterial resistance develops, poor pharmacokinetics and accumulation in urine.8,10-12 However, the NA scaffold is a useful starting point for the design and development of new quinolones with improved bioavailability and tissue penetration.12 The modification of quinolones with synthetic appendages such as peptides may offer an alternative strategy to improve the drug resistance. The conjugation of antibacterial drugs to synthetic vectors such as peptides or carbohydrates can significantly improve their physiochemical properties as well as inter- and intracellular distribution, presenting a means to optimize antibacterial activity.13-17 Attachment of drugs to cell-penetrating peptides (CPPs) has been shown to improve cellular uptake and antibacterial efficacies. Wong and coworkers prepared aminoglycosides-CPP conjugates, which exhibited a 10,000-fold increase in potency in persistent bacteria, as compared to drug alone.13 Our group has developed a family of peptides that target mitochondria, and we have also shown that these sequences are efficient bacterial transporters.16-20 These peptides were designed to improve drug bioavailability and targeting efficacies in both gram-positive and gram-negative bacteria, whereas the drug was unavailable to cytosolic targets in human cells because of sequestering into the mitochondria of mammalian cells, hence making the conjugates inactive in mammalian cells.16-17 Here, we describe a novel approach to modulate the biochemical activity of Nalidixic acid via the incorporation of cationic and hydrophobic peptides. The combination of small peptides (3-12 amino acids) of varying hydrophobicity and net positive charge with the NA core significantly enhanced antibacterial properties and DNA gyrase inhibition efficacies. The peptide/drug conjugates developed with a finely balanced combination of hydrophobicity and molecular charge led to the development of potent NA conjugates with improved DNA


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gyrase inhibition. This study highlights that subtle modulation of the physical properties of the drug significantly improved its topoisomerase inhibition profile.

RESULTS AND DISCUSSION Nalidixic acid is a reversible topoisomerase II inhibitor that selectively interacts with the DNA gyrase of gram-negative. Modification of the drug with electron-withdrawing or electron-donating small molecules has been shown previously to tune the activity of the drug.12 In this study, modification of NA with peptides of varying hydrophobicity and cationic content is used as a means to improve the uptake, toxicity, and - interestingly - DNA gyrase inhibition in S. aureus. Peptide of varying hydrophobicity and molecular charge were prepared by solid-phase peptide synthesis and conjugated with NA. The chemical structures of these peptide-drug conjugates are shown in Figure 1. The molecular weights of peptides and their corresponding NA conjugates were confirmed by ESI-MS and the purity of conjugates was tested using analytical HPLC (Supporting information Figure S1-S10). The hydrophobicity of the peptide-NA conjugates was precisely tuned by incorporation of hydrophobic residues (cyclohexylalanine, Fx,) within a cationic scaffold of D-arginine (r), and the hydrophobic character of peptide-drug conjugates was profiled by monitoring the elution profiles of the compounds. Conjugation of NA to peptide scaffolds yielded a range of hydrophobicities for the cationic peptide/drug conjugates (Supporting information Table S1). The peptide-NA conjugates were tested for antimicrobial efficacies in both MRSA and MSSA strains of S. aureus. (Table 1) As expected, unconjugated NA showed limited activity towards MRSA and MSSA. Peptide vectors alone did not show significant toxicity to both MRSA and MSSA at all concentrations investigated. However, several of the peptide/drug conjugates, compounds 4 and 5 in particular, exhibited much lower IC50 and MIC values in both strains of the bacteria.


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A comparison of the hydrophobicities and molecular charges of the peptide/drug conjugates indicates that these two properties exert significant influence over the activities of these molecules. Hydrophobicity, measured by monitoring HPLC elution profiles, increased in the following order: 2 10-fold higher for the fibroblasts versus the S. aureus strains tested. This trend indicates that the drug conjugates may possess a suitable therapeutic window for successful application as antibacterial agents.


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Further structural studies will be required to elucidate how the peptideNalidixic acid conjugates inhibit DNA gyrase. Given that the potencies of the conjugates vary significantly according to the exact composition of the appended peptide, it seems likely that the peptide is facilitating interaction of NA with the gyrase active site.

The peptides that produced the most active compounds,

(Fxr)3 and (Fxr)3Fx, are both hydrophobic and carry a net +3 molecular charge. Both of these properties appear important for the activity of the compound, indicating that hydrophobic and electrostatic interactions with DNA gyrase play a role in the antibacterial activity of these compounds. Overall, these studies support the idea that by modifying the NA core with peptides of finely-tuned hydrophobicities and controlled levels of molecular charge, the antibacterial properties of the parent drug could be successfully enhanced. The conjugation of the parent drug to short peptides produced significant enhancement of potency, and could be closely linked to the inhibition of DNA gyrase and interference with bacterial DNA synthesis. The activity of the drug conjugates was not directly modulated by peptide-mediated uptake, and it was also shown that the drug conjugates did not induce membrane permeabilization. While several new quinolone-based inhibitors have been reported, most are generated using complex chemical approaches.5-7,25-29 The approach reported offers an alternative and straightforward method to prepare potent antibacterial compounds exhibiting a well-understood mechanism of action related to the inhibition of DNA gyrase. Another potential application of these compounds relates to the inhibition of human topoisomerases, which is of interest for the development of anticancer therapeutics.30


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ACKNOWLEDGMENTS The authors would like to thank S. Mack for useful discussions about the project. This work was supported by the National Sciences and Engineering Research Council and the Canadian Institutes of Health Research.

ASSOCIATED CONTENT Supporting Information is available including supporting data and methods. This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

METHODS Materials. Peptide synthesis reagents were obtained from Chem Pep Inc. The tissue culture reagents and chemicals were obtained from Sigma Aldrich, unless specified otherwise. Supercoiling assay and cleavage assay kits were obtained from Inspiralis.

Synthesis of Peptide Drug Conjugates and Characterization. Peptides were synthesized via solid-phase peptide synthesis, on rink amide MBHA resins, as described previously.17 Nalidixic acid was conjugated at the N-terminus of FMOC-protected peptide following deprotection with 20% piperidine in N’,N’dimethyl formamide (DMF). Briefly, Nalidixic acid was reacted to the amine of peptide in 2:1 molar ratio, using O-(benzotriazol-l-yl)-N,N,N’,N’-tetramethyl-


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uronium hexafluorophosphate (HBTU) (2X) and N,N-diisopropylethylamine DIPEA (4X) in DMF, overnight. Peptide-Nalidixic acid conjugates were cleaved from resins using trifluoroacetic acid:triisopropylsilane:water (TFA:TIPS:H2O) (95:2.5:2.5 v/v) and were precipitated in ether at -20 °C. The peptide-drug conjugates were purified by RP-HPLC on a preparative C18 column, and were lyophilized to obtain pure product. The identity of the compound was confirmed by electron spray ionization mass spectrometry (ESI-MS) (Supporting Information Figure S1-S5). The stock solutions of known concentrations of peptides were prepared by adding desired amount of peptides in DMSO. The purity of peptide conjugates was tested by analytical RP-HPLC on C18 column, with water acetonitrile gradient, containing 0.1% TFA (Supporting information Figure S6-10). Fluorescent peptides were prepared by addition of Carboxytetramethyl rhodamine (TAMRA, anaspec) at N-terminus of peptides by carbodiimide chemistry. Briefly, deprotected-peptides were treated with TAMRA (2X excess), HBTU (2X excess) and DIPEA (4X excess) in DMF for 2 hours. Fluorescent peptides obtained were cleaved purified by RP-HPLC and lyophilized to obtain pure product. TAMRA labeled peptides were quantified in methanol (MeOH) using extinction coefficient of 5.3 X 104 M-1cm-1. Nalidixic acid-(Fxr)2-CONH2 (3) expected m/z = 852, Found m/z = 849.5. Nalidixic acid-(r)3-CONH2 (2) expected m/z =703 , Found m/z = 699.4. Nalidixic acid-(Fxr)3-CONH2 (4) expected m/z = 1162, Found m/z = 1158.7. Nalidixic acid-(Fxr)3Fx-CONH2 (5) expected m/z = 1315, Found m/z= 1311.9. Nalidixic acid-(FxrAr)3-CONH2 (6) expected m/z =1847 , Found m/z = 1841.1.


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TAMRA-(Fxr)2-CONH2 expected m/z =1050 , Found m/z = 1048. TAMRA-(r)3-CONH2 expected m/z =901, Found m/z = 898. TAMRA-(Fxr)3-CONH2 expected m/z = 1361, Found m/z = 1358. TAMRA-(Fxr)3Fx-CONH2 expected m/z =1514 , Found m/z = 1511. TAMRA-(FxrAr)3-CONH2 expected m/z =2045 , Found m/z = 2039.

Toxicity of Peptide-Drug Conjugates in S. aureus. Methicillin resistant (MRSA) and Methicillin sensitive (MSSA) S. aureus were grown overnight in tryptic soy broth (TSB). MRSA and MSSA are then sub-cultured at 1:100 and are grown to log phase (A600 ~ 0.3). Bacteria are further diluted 1:2000 in fresh media in the presence of varying concentrations of peptides, drugs and peptide drug conjugates for 24 hours at 37 °C. The plates were read at 600 nm and MIC values were calculated.

Uptake of TAMRA labeled Peptides in S. aureus. TAMRA labeled peptides were studied for their uptake in MRSA and MSSA strains. Briefly, overnight cultures of bacteria were diluted 1:100 in tryptic soy broth (TSB) and were grown to log phase. The bacteria in log phase were then treated with 10 µM fluorescent peptide in TSB and were incubated for 60 minutes. The bacteria were then washed with phosphate buffered saline (PBS) were suspended in PBS and were analyzed for the uptake of peptides on a BD FACS Canto flow cytometer (BD Bioscience), using excitation wavelength of 546 nm and emission wavelength of 580 nm.


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Flow Cytometric Analysis of Membrane Permeability of S. aureus. MSSA and MRSA in log phase were treated with different concentrations of test compounds for 2 hours. TOPRO-3 Iodide (Invitrogen) was then added to treated bacterial samples at final concentration of 1 µM. The samples were incubated in the dark for 15 minutes and change in membrane permeability was measured by BD FACS Canto flow cytometer (BD Bioscience) using excitation of 642 nm and emission of 660 nm.

Thymidine Incorporation Assay. MRSA and MSSA were cultured overnight in cation adjusted Mueller Hinton Broth and were diluted 1:100 in defined media. Defined media was prepared as described elsewhere.22 Bacteria were grown to log phase in defined media and were incubated with methyl-tritiated thymidine (Perkin Elmer) in the presence or absence of test compounds for 20 minutes. Bacteria grown in the presence of methyl-tritiated thymidine, and in the absence of test compounds were taken as positive control. The DNA samples were precipitated with ice cold Ethanol : TCA, (95:5 v/v), were filtered using glass microfiber filters (Fisher) and were washed with 5% ice cold TCA. The precipitates containing filters were dried and their radioactivity was measured in Ultima Gold F (Perkin Elmer) scintillation fluid using LS 6500 Multi-Purpose Liquid scintillation counter (Beckman Coulter). Toxicity of Peptide-Drug Conjugates in Fibroblasts. Healthy human fibroblasts were a kind gift from Sondheimer Lab at Sick Kids Hospital. Human


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fibroblasts were maintained in low glucose DMEM media, supplemented with 10% FBS, 1 mM sodium pyruvate and 45 µg/mL uridine and were sub-cultured twice a week. Fibroblasts were seeded in 96 well plates and at about 70% density were treated with varying concentrations of test compounds. The plates were incubated for 24 hours and cell viability was tested using CCK-8 assay, as per manufacturer’s protocol.

Supercoiling activity of Peptide-Drug Conjugates. 0.5 µg of relaxed plasmid DNA was incubated with S. aureus DNA Gyrase for 30 minutes in the presence or absence of test compounds in reaction buffer. The plasmid was extracted with chloroform:isoamyl alcohol (24:1 v/v) and was loaded on 1% agarose gel at ~ 60V. Gel was run for 2 hours, was stained with SyBR Green (Invitrogen) for 45 minutes and was visualized using Gel Doc EZ Imager (Biorad).

DNA Cleavage Assay of Peptide-Drug Conjugates, 0.5 µg of supercoiled plasmid DNA was incubated with S. aureus Gyrase for 2 hours in the presence or absence of test compounds in reaction buffer. The reaction was stopped using proteinase K in SDS buffer. DNA was extracted with chloroform:isoamyl alcohol (24:1 v/v) and was loaded on 1% agarose gel at 60V. Gel was run for two hours, stained with SyBR Green (Invitrogen) for 45 minutes and was visualized using Gel Doc EZ Imager (Biorad).


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O

O

HO

A

N

DNA gyrase

N

Nalidixic acid

NH2+

H 2N HN O

O

HN O

H N

N H

O

NH2+

H 2N

HN

H N

H 2N

NH2+

H 2N

N H

O

O

H N

O

N H

O

N

S. aureus

N

Nalidixic acid/peptide conjugate

B X= O

O

NH2+

H 2N

1 NA

HN

H 2N

N

O

2 (r)3 - NA H 2N

NH2

+

H 2N

HN O H 2N

NH2

+

H 2N

HN O

H N O

N H

5 (Fxr)3Fx - NA

O

N H

O NH +H

NH2

2N

NH2

O

O

N H

O

O

O

O

N H

HN

X

N H

O

O

H N

H 2N

N H

NH2+

H 2N

HN

O

3 (Fxr)2 - NA

HN O

H N

N H

O

H N O

N H

X

4 (Fxr)3 - NA NH2+

NH2+

H 2N

O H 2N O

N H

O

O

N H

O

H N O

N H

NH2+

H 2N

O

H N O

N H

NH2+

H 2N HN

HN

HN

HN

H N

NH2+

H 2N

NH

H 2N

HN

HN

H N

O X

NH2+

H 2N

HN

H N

N H

NH2+

H 2N

HN

H N

H 2N

H 2N

+

HN

H N

X

NH2+

H 2N

HN

H N

N H

NH2+

H 2N

HN O

N

NH2+

H 2N

O

H N O

N H

O

H N O

N H

6 (FxrAr)3 - NA

Figure 1. Enhancing the potency of Nalidixic acid with appended peptides. (A) Characterization of the effects of Nalidixic Acid-peptide conjugates versus the parent compound. (B) Compounds used to test the influence of an appended peptide. ACS Paragon Plus Environment

H N X


3500 Relative Fluorescence Intensity (a.u)

3000 2500

(r)3

Untreated

(Fxr)2

(FxrAr)3

(Fxr)3

(Fxr)3-Fx

2000 1500 1000 500

SS A M

R

SA

0

M

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 20 of 26

Figure 2. Uptake of TAMRA labeled peptides in MRSA and MSSA. Bacteria are incubated with fluorescent peptides for 60 minutes, and are evaluated for relative fluorescence intensity using flow cytometry.


MRSA MRSA

500 500 400 300 200

4X

1X

100 0

IC50

800 800 600 600

400 400 400 400 300 300 200 200 100 100

2500

Relative Fluorescence Intensity (a.u)

1000 1000

(Fxr) acid (Fxr)33-Nalidixic -NA Nalidixic Acid NA (Fxr) (Fxr)33 Untreated Untreated Nisin-treated Nisin Heat-treated Heat Dead

2000 1500 1000 500 400 300 200 100

00

0

2X

1500 1500

2X


2000 2000

1000 1000

1X

(Fxr) acid (Fxr)3-Nalidixic -NA Nalidixic Acid NA (Fxr) (Fxr)3 Untreated Untreated Nisin Nisin-treated Heat Dead Heat-treated


2500 2500

Relative Fluorescence Intensity (a.u.)

MSSA MSSA

Relative Fluorescence Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


4X

Page 21 of 26

IC50

Figure 3. Analysis of membrane permeability of MSSA and MRSA treated with Nalidixic acid (NA), (FxR)3 and (FxR)3-NA conjugates at varying concentrations. Bacterial strains are treated with different compounds for 2 hours and change in membrane permeability is assessed by TO-PRO3-Iodide fluorescence intensity.


12

25

50

100

Concentration (µM)

200

800

12

25

12

NA DNA 25 DNA+

DNA DNA+ Cleaved Gyrase DNA DNA+ Gyrase 0 DNA

6

Nalidixic acid

1.2 1.0

50 Concentration 100 (µM)200 Concentration (µM) (µM) 50 50 200 6 Concentration 12 100 25[NA] µM 100 66

1.2

Nalidixic acid ACS Chemical Biology

Concentration (µM)

A+ 6 ase1 A+ 6 2 Nalidixic acid ase 3Nalidixic acid Nalidixic acid

Relative Density of Relaxed DNA Relative Density of Relaxed DN Relative Density of Relaxed DNA Relative Density of Relaxed DNA Relative Density of RelaxedRelative DNA Relative Density of Relaxed DNA Relative Density Density of Relaxed of Relaxed DNA DNA Relative Density ofDensity Relaxed DNADNA Relative of Relaxed Relative Density of Relaxed DNA Relative Density of Relaxed DNA Relative Relative Density Density of Relaxed of Relaxed DNA DNA Relative Density of Relaxed DNA Relative Density of Relaxed DNA Relative Density of Relaxed DNA Relative Density of Relaxed DNA Relative Density of Relaxed DNA Relative Relative Density Density of Relaxed of Relaxed DNA DNA Relative Relative Density Density of Relaxed of Relaxed DNA DNA Relative Density of Relaxed Relative Density DNA of Relaxed DNA ative Density of Relaxed DNA Relative DensityRelative of Relaxed DNA Relaxed DNA Density of Relaxed DNA ty of Relaxed DNA Relative Density ofRelative Density of Relaxed DNA

yrase 6

12

1212

25

2525

50

5050

100

100 100

800

800 200

200

200 200

Page 22 of 26

1.0 0.8

800

800

800 800

0.8 0.6 0.6 0.4 0.4 0.2

Nalidixic acid

1.2

1.0 1.2 1.2

1.2 0.8

NalidixicNalidixic acid Nalidixic acid Nalidixic acid

acid

Relative Density of Relaxed DNA

Gyrase 1.0 4 Concentration (µM) 0.01.2 0.2 1.0 1.0 0.60.80.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 5 1.0 0.0 0.8 0.8 DNA DNA+ 6 12 25 50 100 200 800 0.40.6 Log Concentration (uM) 6Nalidixic acid 0.0 0.8 0.6 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Gyrase 0.6 0.4 Nalidixic acid 0.6 7 1.2 0.4 0.2 Log Concentration (uM) 0.2 0.4 1.0 0.4 8 0.2 0.0 NA+ 1.5 3 6 12.5 25 50 100 200 0.0 0.8 0.2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.2 9 0.0 0.5(FXR) 1.0 1.5-Nalidixic 2.0 2.5 3.0 acid 3.5 rase 0.6 0.00.0 0.5 1.0 1.5 2.03 2.5 3.0 3.5 1.20.0 0.5 1.0 Log 3 6 12.5 25 50 100 200 101.5 Concentration (uM) 1.5Concentration 2.0 2.5(uM) 3.0 (uM) 3.5 Log 0.0 Log Concentration 0.4 (FXR) 0.0Concentration 0.5 3-Nalidixic 1.0 2.0 2.5 3.0 3.5 11 1.0 Log (uM) 1.5 acid 1.2 0.2 12 Log Concentration (uM) 0.0 0.8 [(Fxr) -NA] µM (F r) -NA DNA DNA+ 1.5 3 66 12.5 100100 200 200 DNA+ 1.5 12.5100 1.0 3 2525 50 50 200 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 acid + (Fxr) 1.5 3x 3 DNA 6 12.5 25 50 acid 13(Fxr) 3-Nalidixic 3-Nalidixic Cleaved (FXR)3(FXR) -Nalidixic acid DNA Gyrase DNA+ 1.5 3 6 12.5 25 50 100 200 Gyrase 0.6 3-Nalidixic acid Concentration (uM) 0.8 1.2Log1.2 1.5 3 6 12.5 25 50 100 200 0 (FXR) se 14(Fxr)3-Nalidixic acid (FXR)3-Nalidixic acid 3-Nalidixic acid DNA Gyrase 0.41.2 1.2 1.0 1.0 0.6 + 15 1.5 3 6 12.5 25 50 100 200 0.21.0 0.81.0 DNA DNA+ 1.5 3 6 12.5 25 50 100 200 0.8 (FXR)3-Nalidixic acid e 16(Fxr)3-Nalidixic acid 0.4 0.8 Gyrase 0.6 (FXR) 1.2 3-Nalidixic acid 0.0 0.6 0.8 1.2 17 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.2 0.6 0.4 Figure 4. Inhibition of the activity of S. 1.0 1.0 0.60.4 18 0.4 Log Concentration (uM) 0.2 0.0 0.8 0.2 aureus DNA gyrase in the presence of 0.8 0.2 19 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.00.4 0.6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.0 peptide-Nalidixic acid conjugates, the 20 Log 0.0 0.5 Concentration 2.03.02.53.5 3.0 (uM) 3.5 0.20.6 0.4 0.0 0.5 1.51.0 2.01.52.5(uM) Log1.0 Concentration 21 Log Concentration parent compound, and an unconjugated Log Concentration (uM) (uM) 0.2 0.00.4 DNA+ 6 12 25 50 100 200 400 800 22 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 (FxrAr)3-Nalidixic acid peptide. Inhibition of supercoiling of DNA by 0.0 0.5 0.2 1.0 1.5 2.0 2.5 3.0 3.5 Gyrase (FxRAR)3-Nalidixic acid (FxrAr) acid (FxrAr) acid 23 3-Nalidixic 3-Nalidixic Log Concentration (uM) 1.2 Log Concentration (uM) DNA DNA+ 6 12 25 50 100 200 400 800 (F12 (FxR)3-Nalidixic acid, Nalidixic acid, and xrAr)3-NA [(F -NA] µM 400 0.0 25DNA 50 10012 200 xRAR) 24 6 DNA DNA+ DNA+ 2525 5050 3 400 100100 200200800 Gyrase 66 12 4008 800 800 Cleaved 0.0(FxRAR) 0.53-Nalidixic 1.0 acid 1.5 2.0 2.5 3.0 3.5 1.0 1.2 (FxR)3 was measured using wildtype S. Gyrase (FxRAR) acid (FxRAR) acid acid 25 (FxrAr)3-Nalidixic acid (FxRAR) 3-Nalidixic DNA Gyrase 3-Nalidixic 0 6 12 25 50 100 200 400 3-Nalidixic 1.2 1.2 8 Log Concentration (uM) 0.8 1.0 1.2 26 aureus DNA gyrase. Agarose gel DNA DNA+ 6 12 25 50 100 200 400 8 800 8 8 1.0 1.0 0.8 1.0 0.6 NA+ 6 12 25 Gyrase 50 100 200 400 800 (FxRAR) -Nalidixic acid 27 3 electrophoresis images were quantified and 0.6 1.2 0.8 0.4 0.8 0.8 rase (FxRAR)3-Nalidixic acid 28 8 1.0 0.6 0.4 0.6 relative densities of relaxed DNA were 1.2 0.2 0.6 29 0.8 0.4 0.2 0.4 A+ 6 12 25 50 100 200 400 8008 1.0 calculated to yield the IC50 values listed in 0.2 0.0 30 0.60.4 0.0 ase 0.2 acid 0.0 1.5 3.0 2.0 2.5 3.0 3.5 0.0 0.5 0.5 1.0 1.51.0 2.0(FxRAR) 2.5 3.5 3-Nalidixic 0.0 0.8 0.4 31 Table 1. Additional trials are shown in 1.2 1.5 2.0 2.5 3.0 3.5 0.20.0 0.0 0.5 Log1.0 Concentration (uM) Log Concentration (uM) 0.2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 32 0.6 8 Log Concentration (uM) Figure S1. 1.0 0.0 0.0 Log Concentration (uM) 2.5 3.0 3.5 33 1.53.5 2.0 0.0 0.0 0.5 0.4 1.00.5 1.5 2.01.0 2.5 3.0 (Fxr) 0.8 Log Concentration (uM) 34(Fxr)33 DNA DNA+ 1.5 3 6 12.5 25 50 100 200 Concentration (uM) 0.2 Log 0.6 (FXR)3 NA+ 1.5 3 6 12.5 25 50 100 200 DNA DNA+ 1.5 3 6 12.5 25 50 100 200 Gyrase (Fxr) 35 3 1.2 (FXR) (FXR) (F r) 3 Gyrase 3 1.5 3 6 [(Fxr) 12.5 3] 25 100 200 0.0 rase µM 50 xDNA3 DNA+ 1.2 1.0 1.2 0.4 36(Fxr)3 0.0 0.5 (FXR) 1.03 1.5 2.0 2.5 3.0 3.5 DNA Gyrase DNA+ 1.5 3 6 12.5 25 50 100 200 Cleaved 1.2 1.0 0.8 (FXR)3 37 1.5 3 6 12.5 25 50 100 200 0 1.0 Gyrase 0.2 Log Concentration (uM) 1.2 0.8 1.0 1.5 3 6 DNA12.5 25 50 100 200 0.6 38 (FXR)3 0.8 1.0 0.6 0.0 0.4 0.8 1.2 39 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.8 0.6 0.4 0.2 0.6 40 0.61.0 Log Concentration (uM) 0.2 0.4 0.4 0.0 + 41 1.5 3 6 12.5 25 50 100 200 0.40.8 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 (FXR)3 0.20.0 0.2 se 0.5 1.5 2.0 2.5(uM) 3.0 3.5 0.2 Log1.0 Concentration 42 0.6 1.2 0.0 Log Concentration (uM) 0.0 0.0 0.00.5 1.02.51.53.0 2.5 3.02.5 3.5 3.0 3.5 43 1.0 0.0 0.5 1.0 1.50.5 2.01.0 0.0 1.52.03.5 2.0 0.4 + 3 6 12.5 25 50 100 200 Log Concentration (uM) Log Concentration (uM) 44 1.5 Log Concentration (uM) (FXR) 0.8 3 0.2 e 1.2 45 0.0 0.6 46 ACS Paragon Environment 0.0 1.0 0.5 Plus 1.0 1.5 2.0 2.5 3.0 3.5 0.4 47 0.8 Log Concentration (uM) 0.2 48 0.6

40 30 20 10

(Fxr)3

100 80 60 40 20

0

0

80

80

0

0

NA

IC50 (µM)

0

50

120

80

NA (Fxr)3

(Fxr)3-NA

0

60

140

80

(Fxr)3-NA

MRSA

160

7

80

% Tritiated Thymidine Incorporation

MSSA

160

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


% Tritiated Thymidine Incorporation

Page 23 of 26

IC50 (µM)

Figure 5. Inhibition of DNA synthesis in treated bacteria. MRSA and MRSA are treated with (Fxr)3, (Fxr)3Nalidixic acid and Nalidixic acid for 20 minutes in the presence of tritiated thymidine. The presence of tritium in treated and untreated samples of bacteria is measured by liquid scintillation counting.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 24 of 26

Table 1: IC50 and MIC values for peptide-Nalidixic Acid conjugates in methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains of Staphylococcus aureus, and for the S. aureus DNA gyrase. (*) indicates the conjugates are non-toxic at all studied concentrations. Compound

IC50 (µM) MSSA

IC50 (µM) MRSA

MIC (µg/mL) MSSA

MIC (µg/mL) MRSA

IC50 (µM) S. aureus DNA gyrase

Nalidixic Acid (1)

101 ± 12

152 ± 4

47

47

800

(r)3-NA (2)

> 80

> 80

n.d*

n.d*

> 800

(Fxr)2 – NA (3)

52 ± 1

57 ± 0.8

74

68

800

(Fxr)3 – NA (4)

6.0 ± 3.3

7.0 ± 1.4

18

18

25

(Fxr)3Fx – NA (5)

7±2

6.0 ± 0.1

13

13

25

(FxrAr)3 – NA (6)

20 ± 2

61 ± 2

37

147

200

(Fxr)3

> 80

50 ± 1

n.d*

n.d*

N/A

(FxrAr)3

> 80

> 80

n.d*

n.d*

N/A

Table 2: IC50 values for peptide-Nalidixic Acid conjugates and components tested with human fibroblasts Compound Nalidixic Acid (1) (Fxr)3 – NA (4) (Fxr)3

IC50 (µM) Fibroblasts, strain 1 > 80

IC50 (µM) Fibroblasts, strain 2 > 80

> 80

> 80

102 ± 3

78 ± 5


Page 25 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


TOC graphic

O

O

S. aureus

IC5 > 0 100 µ M

HO N

NH2+

H 2N

O H 2N

NH2+

H 2N

HN O

O

N H

NH2+

H 2N

HN

H N

N

< IC 50

HN O

H N O

N H

O

H N O

10 µ

O

M

DNA gyrase

N H N

N



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC graphic 881x661mm (72 x 72 DPI)


Page 26 of 26

Enhancing the Potency of Nalidixic Acid toward a Bacterial DNA

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