Bromophenazine derivatives with potent inhibition, dispersion and eradication activities against Staphylococcus aureus biofilms

Abstract

Bacterial biofilms are surface-attached communities of bacteria that are: (1) highly prevalent in human infections, and (2) resistant to conventional antibiotic treatments and host immune responses. It has only been in the last ∼20 years that bacterial biofilms have been identified as a critical biomedical hurdle in infectious disease and human health. Staphylococcus aureus is a leading cause of nosocomial and community-acquired infections and is notorious for its ability to form drug-resistant biofilms. Despite the need for antibacterial agents that target S. aureus biofilms, few chemical scaffolds are known that are capable of inhibiting, dispersing or eradicating their biofilms. Here, we report the discovery of bromophenazine derivatives that display antibiofilm activities as either potent biofilm inhibitors (IC₅₀ values 0.55–10.3 μM) or dispersal agents (EC₅₀ values 1.4–29.3 μM) and biofilm eradicators (MBEC values 100–200 μM) against S. aureus strains, including a methicillin-resistant Staphylococcus aureus clinical isolate. These discoveries could lead to the development of new treatment options that target drug-resistant, biofilm-associated S. aureus infections.

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Staphylococcus aureus is a Gram-positive human pathogen that is notorious for its role as a leading cause of both nosocomial and community-acquired bacterial infections worldwide.^1,2 During infection, S. aureus forms surface-attached bacterial communities, known as biofilms, that are intrinsically resistant to conventional antibiotic treatments and host immune responses.^2,3 As a result of the innate antibiotic resistance displayed by these surface-attached bacterial communities, S. aureus biofilms are essentially impossible to clear or eradicate in the clinic.³

Unfortunately, our current arsenal of antibiotics does not hit bacterial targets critical to biofilm formation or maintenance. Despite the unmet biomedical challenge posed by biofilm-associated bacterial infections, many pharmaceutical companies have eliminated their antibacterial discovery programs.^4–6 Future antibacterial agents will require the ability to modulate biofilm processes, such as quorum sensing, biofilm formation and maintenance⁷ or eradicate established biofilms.^8,9

S. aureus biofilm-related diseases are highly prevalent in osteomyelitis, indwelling medical device infection, periodontitis and peri-implantitis, chronic wound infection, chronic rhinosinusitis, endocarditis, ocular infection and polymicrobial biofilm infections.² Despite the overwhelming need for therapeutic options to treat biofilm-associated S. aureus infections, few small molecule scaffolds have been reported that target these biofilms³ (e.g., ADEP4 (ref. 10), cis-2-decenoic acid,¹¹ 2-aminoimidazoles,^12,13 aryl rhodanines,¹⁴ quinazolinones¹⁵). Antibiofilm agents that are clinically effective against S. aureus biofilms will have a significant impact on the treatment of drug-resistant, biofilm-associated S. aureus infections.

Our group recently identified bromophenazine 1 (Fig. 1) as a potent antibacterial agent against S. aureus (minimum inhibitory concentration or MIC 1.56 μM) inspired by the redox-active phenazine antibiotic pyocyanin that is produced by Pseudomonas aeruginosa.¹⁶ Pyocyanin causes oxidative stress in various cell lines and is associated with toxicity;¹⁷ however, despite these concerns, pyocyanin has promising pharmacological applications.¹⁸ Pyocyanin, in part, enables P. aeruginosa to clear cystic fibrosis patients' lungs of established S. aureus infection during competitive microbial interactions within the lung.¹⁹ In this study, we were interested in investigating the potential to clear biofilm-associated S. aureus infections with bromophenazine small molecules. With the growing demand for effective antibiofilm agents against S. aureus biofilms, we were interested in synthesizing and evaluating derivatives of 1 in a series of biofilm inhibition, dispersion and eradication assays against this major pathogen.

Fig. 1 Bromophenazine 1 is a potent antibacterial agent and potentially a new platform to target S. aureus biofilms.

Bromophenazine derivatives were synthesized by reacting 1 with commercially available acid chloride or chloroformate starting materials in chloroform with 4-(dimethylamino)pyridine (DMAP) as a catalyst (Scheme 1). These reactions typically required stirring for one hour at room temperature to give an average yield of 86% (range: 65 to >99% yield) for bromophenazines 2–11 following column chromatography purification. Following the synthesis of each bromophenazine derivative (1–11), DMSO stock solutions were prepared for biological evaluation in MIC, biofilm inhibition, biofilm dispersion and biofilm eradication experiments against S. aureus strains (including ATCC 25923 and MRSA-2, a methicillin-resistant Staphylococcus aureus clinical isolate²⁰) in 96-well plates.

Scheme 1 Chemical synthesis of bromophenazine derivatives 2–11.

We began our biological investigations of bromophenazines 1–11 against S. aureus by performing two series of assays against S. aureus ATCC 25923, which included: (1) microdilution MIC experiments to evaluate planktonic growth inhibition activity and (2) biofilm inhibition assays using crystal violet staining to quantify S. aureus biofilm formation. Initially, we wanted to have a single assay that would allow us to obtain biofilm inhibition data directly from MIC experiments (i.e., 16–20 h incubation at 37 °C, ∼10⁵ CFU mL⁻¹, Luria–Bertani medium) against S. aureus; however, alternative assay conditions were necessary to have optimal biofilm formation in 96-well plates (i.e., gelatin pre-coated wells, 24 hours at 37 °C, ∼1 × 10⁶ CFU mL⁻¹, tryptic soy broth medium with 0.5% glucose). These assay conditions resulted in a robust S. aureus biofilm and served as an excellent model for our investigations.

In general, compounds that inhibit biofilm formation through non-growth inhibiting mechanisms are considered true biofilm inhibitors whereas compounds that inhibit bacterial growth while inhibiting biofilm formation are considered to demonstrate an antibacterial phenotype.²⁰ Biofilm-inhibiting small molecules prevent bacterial biofilm formation without placing a selective pressure on bacteria to develop resistance.³ Bromophenazine derivatives 4, 5, 6 and 7 demonstrated potent “biofilm inhibition” activity against S. aureus without demonstrating planktonic growth inhibition (Fig. 2A for biofilm inhibitors 4 and 5; Table 1).

Fig. 2 (A) Planktonic growth and biofilm inhibition assay with 4 and 5; (B) biofilm dispersion assays with bromophenazines 1, 2, 8, and 9 against S. aureus; (C) bromophenazine 1 potently disperses established MRSA-2 biofilms (EC₅₀ = 3.53 μM).

Table 1 Antibiofilm activity of bromophenazine derivatives 1–11 against Staphylococcus aureus (ATCC 25923^a and MRSA-2^b)

Compound	Growth inhibition MIC^a (μM)	Biofilm inhibition IC50^a (μM)	Biofilm dispersion EC50^a (μM)	Biofilm eradication MBEC^b
a S. aureus ATCC 25923.b MRSA-2. IC50 values reported for a single replicate screen except for 4–7 which are reported from 3 independent biofilm inhibition experiments following the initial screen. EC50 (biofilm dispersion) and MBEC (biofilm eradication) values are reported from 2 to 4 independent experiments.c Bromophenazine 1 gave an EC50 value of 3.53 μM in MRSA-2 dispersion assays (not reported here).d MRSA-2 is “sensitive” to vancomycin (MIC 0.78 μM) as a growth inhibitor.^20
1	1.56^a^,^b	0.41	29.3	100–200^c
2	1.56	0.92	2.6	125
3	1.56	0.76	>100	—
4	>100	0.55	>100	—
5	>100	10.3	>100	—
6	>100	0.77	>100	—
7	>100	1.13	>100	—
8	0.78	0.76	1.4	62.5–100
9	1.56	0.77	2.9	250
10	0.78	0.76	>100	125
11	>100	>100	>100	—
Vancomycin	—	—	—	>2000^d
QAC 10	—	—	—	62.5–125

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The four bromophenazine derivatives (i.e., 4–7) that demonstrated potent S. aureus biofilm inhibition possess a 4-substituted phenyl ester moiety. These derivatives demonstrated sub-micromolar biofilm inhibition activity (IC₅₀ values in biofilm inhibition assays) in our initial single replicate screen against S. aureus ATCC 25923. Final IC₅₀ values for 4–7 were determined by three independent biofilm inhibition experiments and biofilm inhibitor potency ranged between 0.55–10.3 μM (Table 1; ESI†). Bromophenazine 4 demonstrated the most potent biofilm inhibition activity against S. aureus ATCC 25923 with an IC₅₀ value of 0.55 μM (compared to an MIC > 100 μM) to give an MIC : IC₅₀ value ratio of >181 (Fig. 2A.). Bromophenazine 4 is one of the most potent biofilm inhibitors to be reported against S. aureus (IC₅₀ = 550 nM).¹²

The three other bromophenazine “biofilm inhibitors” (derivatives 5–7) report MIC : IC₅₀ value ratios of >10 to >130 in comparison to bromophenazines that demonstrated “antibacterial” activity (i.e., derivatives 1–3; 8–10) that report MIC : IC₅₀ ratios of 1.0 to 3.8 during these investigations against S. aureus (Table 1). We recently reported several antibiotics (i.e., vancoymycin, ciprofloxacin, erythromycin) to have MIC : IC₅₀ value ratios between 3.1 and 7.8 against S. aureus ATCC 29213 in biofilm inhibition assays to reference “antibacterial” activity.²⁰ In the same study, our group identified two biofilm inhibitors that had MIC : IC₅₀ ratios of >22 against S. aureus ATCC 29213.²⁰ Previously, 2-aminobenzimidazole biofilm inhibitors have been reported with MIC : IC₅₀ value ratios that range between 4 to >20.²¹ As part of the discussion to differentiate “biofilm inhibition” activity from “antibacterial” activity, a norspermidine analogue was recently reported to possess biofilm inhibition activity that had no observable growth inhibitory activity until concentrations were 40-fold higher than the concentration of this compound's minimum biofilm inhibitory concentration against B. subtilis.²²

Five of the remaining six bromophenazine derivatives (i.e., 1–3; 8–10) demonstrated antibacterial activity (MIC 0.78–1.56 μM) while bromophenazine 11 demonstrated neither antibacterial nor biofilm inhibition activity at the concentrations tested. Despite potent antibacterial activities against S. aureus, bromophenazines demonstrate weak antibacterial activity against Gram-negative bacteria. Against A. baumannii, bromophenazine 1 gave an MIC 50 μM (ESI†) and an MIC > 100 μM against P. aeruginosa (PAO1).¹⁶

We evaluated our bromophenazine derivatives in biofilm dispersion assays to determine if these bromophenazines were capable of dispersing, or clearing, established S. aureus biofilms. In biofilm dispersion assays, bacterial biofilms are established in 96-well plates in the absence of test compound. Following the establishment of biofilms, media/planktonic bacteria are removed and test compound is added (in buffer or media) to the established biofilms inside microtiter wells and allowed to incubate. Biofilm dispersion is quantified via crystal violet staining of treated biofilms with the use of a spectrophotometer (OD₅₄₀).

We established S. aureus biofilms in 96-well plates using conditions similar to biofilm inhibition assays (ESI†). Following the initial S. aureus biofilm establishment, bromophenazine derivatives 1–11 were added to 96-well plates in 2-fold serial dilutions with established S. aureus biofilms in either: (1) phosphate buffered saline (PBS) with room temperature incubation for 24 hours with established S. aureus ATCC 25923 biofilms or (2) media with 37 °C incubation for 24 hours with MRSA-2 biofilms. Following the final incubation of established S. aureus biofilms with our bromophenazine derivatives, crystal violet was used to stain and quantify remaining biofilms to determine biofilm dispersal activity of bromophenazines 1–11 as effective concentrations from our test concentrations (EC₅₀ values; Table 1).

We identified four bromophenazine derivatives (e.g., 1, 2, 8 and 9; Fig. 2B; Table 1) capable of dispersing established S. aureus ATCC 25923 biofilms. The potency of these four biofilm dispersion-active bromophenazines ranged in EC₅₀ values between 1.4 and 29.3 μM while the three most potent dispersal agents gave EC₅₀ values of 1.4 μM, 2.6 μM and 2.9 μM for derivatives 8, 2 and 9 respectively. Despite several potent antibacterial or biofilm inhibitors, many bromophenazine derivatives were found to be completely inactive in head-to-head dispersion assays against established S. aureus ATCC 25923 biofilms (EC₅₀ > 100 μM). Bromophenazine 1 effectively dispersed MRSA-2 biofilms and reported an EC₅₀ value of 3.53 μM (Fig. 2C).

Since the most potent antibacterial bromophenazines demonstrated a tendency to be potent biofilm dispersal compounds, we wanted to evaluate bromophenazines 1, 2, 8, 9 and 10 in biofilm eradication assays^8,9,23 against MRSA-2 to see if our active compounds were demolishing MRSA biofilms. Biofilm eradication assays are essentially biofilm dispersion assays with the addition of a final treatment with fresh media (and incubation at 37 °C for 24 hours) instead of crystal violet staining. This final incubation allows viable cells within the biofilm to grow. At the end of this final incubation in biofilm eradication assays, microtiter wells void of turbidity represent eradicated biofilms and the lowest concentration at which no visible growth is observed is referred to as the minimum biofilm eradication concentration (MBEC). Potent biofilm-eradicating small molecules are extremely rare.^8,9 For these experiments, we selected MRSA-2 as our model since it is a multidrug-resistant, biofilm-forming clinical isolate of S. aureus.¹⁸ Against MRSA-2, bromophenazine 1 reported an MIC value of 1.56 μM and an MBEC of 100–200 (Fig. 3) while 2, 8, 9 and 10 gave MBEC values between 62.5 and 250 μM. Only bromophenazine 8 was found to be more potent (MBEC 62.5–100 μM) than parent compound 1. Bromophenazine 8 was found to be equipotent to known biofilm eradicating agent⁹ QAC 10 in head-to-head eradication assays against MRSA-2 biofilms.

Fig. 3 Biofilm eradication of MRSA-2 with bromophenazine 1 (MBEC = 100–200 μM) and vancomycin (MBEC > 2000 μM).

We also evaluated vancomycin against MRSA-2 since it is considered to be the drug of last resort against MRSA infections. Vancomycin gave an MIC of 0.78 μM against MRSA-2 and an MIC of 0.39–0.78 against S. aureus ATCC 29213, therefore MRSA-2 is considered to be “sensitive” to vancomycin.²⁰ When tested against MRSA-2 in biofilm eradication assays, vancomycin reported an MBEC of >2000 μM (inactive at all concentrations). The MRSA-2 biofilms are >2564-fold more resistant against vancomycin when compared to their planktonic counterparts (i.e., MBEC : MIC ratio). Bromophenazine 1 reported an MBEC : MIC ratio of 64–128.

We also tested the stability of several ester derivatives towards hydrolysis by subjecting these compounds to conditions mimicking our biofilm assays. All bromophenazines tested were found to be completely stable to hydrolysis (ESI†).

In conclusion, we have discovered several bromophenazine derivatives that are potent inhibitors, dispersal agents and eradicators against S. aureus biofilms, including a MRSA clinical isolate. These bromophenazines are inspired by pyocyanin, thus future investigations to determine the therapeutic index of these compounds are critical. Bromophenazine 1 is a promising small molecule platform to develop agents that target S. aureus biofilms. These findings could lead to breakthroughs in the treatment of drug-resistant, biofilm-associated S. aureus infections worldwide.

Acknowledgements

This work was supported by a Research Opportunity Seed Fund Award and the College of Pharmacy at the University of Florida. We would like to thank Christian Melander for useful comments during the course of these studies. A.T.G. has been awarded a UF Graduate School Fellowship.

Notes and references

1. J. M. Yarwood, D. J. Bartels, E. M. Volper and E. P. Greenberg, J. Bacteriol., 2004, 186, 1838.
2. N. K. Archer, M. J. Mazaitis, J. W. Costerton, J. G. Leid, M. E. Powers and M. E. Shirtliff, Virulence, 2011, 2, 445.
3. P. Y. Chung and Y. S. Toh, Pathog. Dis., 2014, 70, 231.
4. K. Lewis, Nat. Rev. Drug Discovery, 2013, 12, 371.
5. S. J. Projan, Curr. Opin. Microbiol., 2003, 6, 427.
6. J. F. Barrett, Curr. Opin. Microbiol., 2005, 8, 498.
7. D. J. Musk and P. J. Hergenrother, Curr. Med. Chem., 2006, 13, 2163.
8. M. H. Fletcher, M. C. Jennings and W. M. Wuest, Tetrahedron, 2014, 70, 6373.
9. M. C. Jennings, L. Ator, T. J. Paniak, K. P. C. Minbiole and W. M. Wuest, ChemBioChem, 2014, 15, 2211.
10. B. P. Conlon, E. S. Nakayasu, L. E. Fleck, M. D. LaFleur, V. M. Isabella, K. Coleman, S. N. Leonard, R. D. Smith, J. N. Adkins and K. Lewis, Nature, 2013, 503, 365.
11. D. G. Davies and C. N. H. Marques, J. Bacteriol., 2009, 191, 1393.
12. R. W. Huigens III, S. A. Rogers, A. T. Steinhauer and C. Melander, Org. Biomol. Chem., 2009, 7, 794.
13. Z. Su, L. Peng, R. J. Worthington and C. Melander, ChemMedChem, 2011, 6, 2243.
14. T. J. Opperman, S. M. Kwasny, J. D. Williams, A. R. Khan, N. P. Peet, D. T. Moir and T. L. Bowlin, Antimicrob. Agents Chemother., 2009, 53, 4357.
15. Y. Ma, Y. Xu, B. D. Yestrepsky, R. J. Sorenson, M. Chen, S. D. Larsen and H. Sun, PLoS One, 2012, 7, e47255,  DOI:.
16. N. V. Borrero, F. Bai, C. Perez, B. Q. Duong, J. R. Rocca, S. Jin and R. W. Huigens III, Org. Biomol. Chem., 2014, 12, 881 .
17. H. Ran, D. J. Hassett and G. W. Lau, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 14315–14320.
18. P. Priyaja, P. Jayesh, R. Phillip and I. S. Bright Signh, Cytotechnology, 2014 DOI:10.1007/s10616-014-9765-5.
19. Z. A. Machan, T. L. Pitt, W. White, D. Watson, G. W. Taylor, P. J. Cole and R. Wilson, J. Med. Microbiol., 1991, 34, 213.
20. Y. Abouelhassan, A. T. Garrison, G. M. Burch, W. Wong, V. M. Norwood IV and R. W. Huigens III, Bioorg. Med. Chem. Lett., 2014, 24, 5076.
21. E. A. Lindsey, C. M. Brackett, T. Mullikin, C. Alcaraz and C. Melander, ChemMedChem, 2012, 3, 1462.
22. T. Böttcher, I. Kolodkin-Gal, R. Kolter, R. Losick and J. Clardy, J. Am. Chem. Soc., 2013, 135, 2927.
23. Biofilm eradication assays adopted from: (a) Y.-J. Eun, M. H. Foss, D. Kiekebusch, D. A. Pauw, W. M. Westler, M. Tanbichler and D. B. Weibel, J. Am. Chem. Soc., 2012, 134, 11322; (b) H. Ceri, M. E. Olson, C. Stremick, R. R. Read, D. Morck and A. Buret, J. Clin. Microbiol., 1999, 37, 1771.

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Footnotes

† Electronic supplementary information (ESI) available: Synthetic procedures, NMR spectra for new compounds synthesized, MIC, biofilm inhibition/dispersion/eradication protocols, dose–response curves and images of MIC/biofilm experiments. See DOI: 10.1039/c4ra08728c

‡ These authors made an equal contribution to this work.

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