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Antibiotic-derived molecular probesfor bacterial imaging

Mark A. Blaskovich, Wanida Phetsang, M. Rhia L. Stone,Urszula Lapinska, Stefano Pagliara, et al.

Mark A. Blaskovich, Wanida Phetsang, M. Rhia L. Stone, Urszula Lapinska,Stefano Pagliara, Rajiv Bhalla, Matthew A. Cooper, "Antibiotic-derivedmolecular probes for bacterial imaging," Proc. SPIE 10863, PhotonicDiagnosis and Treatment of Infections and Inflammatory Diseases II, 1086303(7 March 2019); doi: 10.1117/12.2507329

Event: SPIE BiOS, 2019, San Francisco, California, United States

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Antibiotic-derived molecular probes for bacterial imaging Mark A. T. Blaskovich*a, Wanida Phetsanga, M. Rhia L. Stonea, Urszula Lapinskab, Stefano

Pagliarab, Rajiv Bhallac, Matthew A. Coopera aInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia

bLiving Systems Institute, University of Exeter, Exeter EX4 4QD, UK cCentre for Advanced Imaging, The University of Queensland, Brisbane, QLD 4072, Australia

ABSTRACT

Infections caused by drug resistant bacteria poses a significant threat to global human health, with predicted annual mortality of 10 million by 2050. While much attention is focused on developing better therapies, improving diagnosis would allow for rapid initiation of optimal treatment, reducing unnecessary antibiotic use and enhancing therapeutic outcomes. There are currently no whole body imaging techniques in clinical use that are capable of specifically identifying bacterial infections. We have developed antibiotic-derived fluorescent probes that bind and illuminate either Gram-positive or Gram-negative bacteria with high specificity and selectivity over mammalian cells. Antibiotics are functionalised with an azide substituent in a position that minimises effects on antibiotic activity. These are reacted by facile 1,3-dipolar cycloaddition with alkyne-substituted imaging components such as visible or near-infrared fluorophores. The resulting adducts can be used as tools to image bacteria in vitro and in vivo. We have successfully functionalised representatives of seven major antibiotic classes. These derivatives retain antibacterial activity, and have been coupled with a range of fluorophores. Fluorescent versions of vancomycin and polymyxin B are particularly useful for specific labelling of G+ve and G-ve bacteria, respectively. Preliminary studies have now extended the visualisation component to include moieties compatible with PET imaging.

Keywords: antibiotics, antibiotic probes, antimicrobial resistance, bacterial imaging, bacterial infection imaging, fluorescent probes, microfluidics, single-cell analysis

1. INTRODUCTION 1.1 Antimicrobial Resistance The increasing incidence of infections caused by drug resistant bacteria and fungi poses a significant threat to global human health, with predicted annual mortality of 10 million by 2050. The United Nations,1 the World Health Organisation,2 the United States Centre for Disease Control and Prevention3 and the Wellcome Trust4 have all highlighted the need for action against this looming menace. The annual health burden in the EU and USA alone has reached $21-34 billion, with nearly 100,000 deaths per year due to antibiotic resistant hospital-acquired infections.5 It is imperative to advance new technologies so that infections are rapidly and accurately diagnosed to improve appropriate therapy, and to develop new antibiotics with efficacy against multidrug-resistant bacteria, supported by fundamental studies on the underpinning chemical biology of antibiotic action and key aspects of bacterial growth, division, metabolism and resistance.

Bacterial sepsis is associated with high rates of mortality (30-80%),6-8 partly due to the length of time required to confirm the etiological agent and initiate the appropriate treatment in a timely manner. Therapy is almost always given empirically at first, which can lead to enrichment of a resistant or persister subpopulation when the wrong antibiotic is given. The gold standard methods for detecting bacteria are still culture based,9, 10 requiring a 24h growth step that leads to long turnaround and analysis times. Rapid detection of bacterial infections would be a major breakthrough to improve infectious disease outcomes, particularly for sepsis and bacteraemia where every minute counts. However, just detecting whether an infection is present is not enough – knowing the source of the infection can be just as important. Determining the tissue type and location of bacterial infections, or assessing whether implants have become infected, often requires invasive techniques. A whole-body imaging diagnostic that could simultaneously determine whether an infection was present and rapidly pinpoint the site of the infection would directly inform targeted treatment. The unmet clinical need for infection imaging diagnostics, and the limitations of current research, are summarised in three recent reviews.4, 11, 12

*m.blaskovich @uq..edu.au; phone 61 (0)7 3346 2994; http://researchers.uq.edu.au/researcher/1614

Invited Paper

Photonic Diagnosis and Treatment of Infections and Inflammatory Diseases II, edited by Tianhong Dai, Jürgen Popp, Mei X. Wu, Proc. of SPIE Vol. 10863, 1086303 · © 2019 SPIE

CCC code: 1605-7422/19/$18 · doi: 10.1117/12.2507329

Proc. of SPIE Vol. 10863 1086303-1Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 09 Mar 2019Terms of Use: https://www.spiedigitallibrary.org/terms-of-use

1.2 Fluorescent Probes Chemical probes, particularly antibiotics,13 are important tools used to unravel complex biological pathways and validate new biological targets.14, 15 Fluorescent probes enable high resolution microscopic imaging to determine probe localization inside biological systems, such as cells, bacteria, and living organisms. There have only been a limited number of antibiotic-based fluorescent probes reported to date: these have proven useful in a range of applications, as summarized in a recent review:16 (i) Antibiotic localisation and MoA studies. Peptidoglycan biosynthesis was studied using vancomycin and ramoplanin fluorescein- and BODIPY-probes,17 while a vancomycin-Oregon Green probe examined delivery of vancomycin into mammalian cells.18 A polymyxin-BODIPY probe visualised the uptake of polymyxin into renal cells.19 Nisin-fluorophore derivatives assessed membrane permeabilisation,20 while a dansylated antimicrobial peptide showed a heterogeneous subcellular localization inside single bacteria cells.21 NBD- and BODIPY- daptomycin probes were recently applied to multiple mode of action studies,22 while a commercial fluorescent penicillin derivative (BOCILLIN-FL) examined drug tolerance in persister bacterial cells.23 ii) Biological target identification and validation. New protein targets of vancomycin were identified using a fluorescent probe.24 Bacterial penicillin binding proteins (PBPs) were labelled with a BODIPY-penicillin derivative, to aid in protein identification,25 while trimethoprim probes labelled intracellular proteins in live cells.26 (iii) Screening assays. BODIPY-erythromycin derivatives were employed for a fluorescence polarisation assay used to identify novel ribosome inhibitors.27 1.3 Imaging of Bacterial Infections Bacterial infection imaging has the potential to 1) discriminate infections from sterile inflammation, 2) visualise the extent of infections, 3) identify difficult to diagnose infections such as infective endocarditis (IE) or prosthetic joint infections (PJIs), and 4) allow for monitoring of therapy effectiveness. Infection imaging must distinguish a pathogenic infection from the normal non-invasive microbiota always present in the body. Research into bacteria-specific imaging is remarkably rare, with only 86 publications between 1988-2015.4 Current clinical imaging techniques largely rely on detecting inflammation associated with infection, with PET or Single Photon Emission Computed Tomography (SPECT) employing non-specific tracers such as [18F]-fluorodeoxyglucose ([18F]-FDG) that do not clearly discriminate between generalised sterile inflammation and infection.11, 12, 28,29 Radiolabeled leukocytes arguably provide greater specificity for infection-related inflammation, but are difficult to prepare and expensive.30

Few bacterial-specific probes have been tested in humans. A [99mTc]-labeled version of ciprofloxacin, Infecton®, was clinically approved for SPECT imaging, but removed from the market due to lack of differentiation between sterile inflammation and infection;11 [18F]ciprofloxacin was also unsuccessful in humans.31 Other reported probes include a [99mTc]-labeled cationic antimicrobial peptide (ubiquicidin, UB129-41)32 and [68Ga]-NOTA-UBI peptide complex,32 while [18F]-Fluorodeoxysorbitol ([18F]-FDS), which selectively accumulates in Gram−ve bacteria, but not in Gram+ve or mammalian cells.33, 34 was examined in a first-in-human evaluation that found no adverse effects, but did not evaluate efficacy.35

At a preclinical research level, bacterial targeting moieties include zinc(II)dipicolylamine,12 bacteriophages,12 and antibodies.4 For example, [64Cu]-DOTA linked to an antibody successfully imaged Aspergillus fumigatus lung infections in mice but took 48 h to distribute.36 [18F]-labeled maltohexaose (MH[18F]), which targets the bacteria-specific maltodextrin transporter, was used for PET imaging of Escherichia coli in rat.37, 38 and a Staphylococcus aureus mock implant infection in rats.39 Similarly, [18F]-fluoromaltose detected E. coli infections in mice,40, 41 with a second-generation 6”-18F-fluoromaltotriose tracer detecting Pseudomonas aeruginosa infection in mice.42 [68Ga]-complexes of DOTA- or NOTA-ciprofloxacin,43 a hybrid UBI complex with [111In],44 [99mTc]-vancomycin,45 and a rhodamine/[125I]-labeled vancomycin-peptide conjugate46 have also been investigated.

2. METHODOLOGY 2.1 Strategy We have developed a research programme in which representatives of major classes of antibiotics are being functionalised with a readily derivatised handle, providing a set of versatile intermediates that can then be further modified to create probes, imaging agents and therapeutics (see Figure 1). Underpinning the synthetic design is a facile


coupling reaction to conjugate the antibiotics with different fluorophores. We have chosen the highly selective “click” Cu(I)-catalysed azide−alkyne cycloaddition (CuAAC) coupling reaction47 as it is high yielding and tolerates the many functionalities present in antibiotics. The azide component resides on the antibiotic as it is synthetically easier to introduce. Alkyne-derivatised fluorophores are readily synthesised. The resulting triazole linkage is well accepted in medicinal chemistry and molecular biology.47 Click chemistry is previously reported for structure-activity studies of antibiotics such as aminoglycosides48 or macrolides,49 but its advantages have not yet been leveraged to prepare multiple colour probe derivatives.

Figure 1. Schematic overview of antibiotic functionalization strategy

In order to minimise disruptions to antibiotic activity and localisation, the fluorophore of the probe must be small, and maintain similar physicochemical properties (e.g. logD) to the parent molecule. Many fluorophores (e.g. rhodamine B) are larger than antibiotics themselves, and hence likely to perturb the interactions being studied. A significant shortcoming of many published antibiotic probe studies is that the biological activity (minimum inhibitory concentration, MIC) of the probe is not reported, potentially invalidating any conclusions. We have focused initially on incorporating green 7-nitrobenzofurazan (NBD) (Figure 2A) and blue 7-(dimethylamino)-coumarin-4-acetic acid (DMACA) fluorophores (Figure 2B).50, 51 Their small size (molecular weight NBD = 164, DMACA = 261) minimises possible interference with activity compared to the commonly used much larger and often charged fluorophores such as Oregon Green (MW = 412) or rhodamine B (MW = 479). Other colours can be accessed through compact BODIPY-based dyes (e.g. Figure 3C), including far-infrared dyes for in vivo imaging.

Figure 2. Fluorophores

2.2 Synthesis and Characterisation Each azide-antibiotic is designed based on published structure-activity relationship data, locating the azide substituent at a position known to be tolerant of substitution. Each new azide-antibiotic is linked to at least two fluorophore-alkynes to create probes with complementary colours. The azide intermediates and assembled fluorescent probes are purified by HPLC, structurally characterised (LCMS, NMR, HRMS), and tested against a panel of bacteria to determine their MIC and confirm that they retain activity. SR-SIM microscopy is employed to visually assess probe interaction with representative bacteria and mammalian cells.


2.3 Minimum Inhibitory Concentration (MIC) determination Bacteria were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), Merck Sharp & Dohm (Kenilworth, NJ), and independent academic clinical isolate collections. Bacteria were cultured in cation-adjusted Muller Hinton broth (CA-MHB) (Bacto laboratories, Cat. no. 211443) at 37°C overnight. A sample of each culture was then diluted 50-fold in CA-MHB and incubated at 37 °C for 1.5-3 h. The compounds were serially diluted two-fold across the wells, with concentrations ranging from 0.06 μg/mL to 128 μg/mL, plated in duplicate. The resultant mid-log phase cultures were diluted to the final concentration of 5 × 105 CFU/mL, then 50 μL was added to each well of the compound-containing 96-well plates (Corning; Cat. No 3641, NBS plates), giving a final compound concentration range of 0.03 μg/mL to 64 μg/mL. All the plates were covered and incubated at 37 °C for 18-24 h with the MIC defined as the lowest compound concentration at which no bacterial growth was visible (n ≥ 4).

2.4 Fluorescence Microscopy: Structured Illumination Microscopy (SIM) SIM was performed using the Elyra PS.1 SIM/STORM microscope. Images were analyzed with ZEN2012. VectaShield H1000 was used as a mounting media. Cover slip glasses (Zeiss/Schott, 18 mm x 18 mm, No.1.5H) were used to prepare samples. Hank’s Balanced Salt Solution (HBSS) without phenol red, CaCl2, and MgSO4 (Sigma Aldrich Cat.-No.: H6648) was used for bacterial staining. Fluorescent dyes FM4-64FX, and Hoechst 33342 (Life Technologies, Australia) were used for membrane staining and DNA staining, respectively. S. aureus (ATCC 25923) and E. coli were cultured in LB at 37 °C overnight. A sample of each culture was then diluted 50-fold in LB and incubated at 37 °C for 1.5-2 h. 1 mL of the resultant mid-log phase cultures were transferred to an Eppendorf tube and centrifuged. Bacteria were washed once with HBSS, then suspended in 20 μL of HBSS. 2 μL of this suspended bacteria solution was dropped onto a cover slip, spread and dried. Probe (200 μL, varying concentrations, e.g. 1-64 μg/mL) was then added to the bacteria, left for 30 min at room temperature, and then washed once with HBSS. For costaining experiments, an ice-cold solution (200 μL) of Hoechst 33342 (5 μg/mL in HBSS) was then dropped onto the bacteria, left for 10 min on ice, then drained. This was followed by adding an ice-cold solution (200 μL) of FM4-64FX (5 μg/mL in HBSS) onto the bacteria, which was left for 5 min for E. coli and 1 min for S. aureus on ice. The bacteria were then washed once with ice-cold HBSS. The bacteria were fixed with 4% paraformaldehyde for 20 min for E. coli and 10 min for S. aureus on ice, followed by mounting on slides using VectaShield H1000 as a mounting media.

3. RESULTS 3.1 Antibiotics Derivatisation and Activity To date, we have prepared azide versions of 8 clinical antibiotic classes – oxazolidinones (linezolid),52 trimethoprim,53 metronidazole, glycopeptides (vancomycin), lipopeptides (polymyxin and daptomycin), fluoroquinolones (cipro-floxacin), and macrolides (roxithromycin), along with three investigational antibiotics – octapeptin, arenicin and tachyplesin. Most of these have then been coupled with two different colour fluorophores, followed by determination of their antibacterial activity (Table 1).

Surface active antibiotics do not need to penetrate inside bacteria to reach their initial target site and are often quite large (MW > 1000) so are more likely to retain activity when modified with fluorophores compared to antibiotics acting on internal targets. Indeed, the azide-functionalised representatives of surface-active antibiotic classes, glycopeptides (vancomycin), lipopeptides (polymyxin B, PmxB; and octapeptin C4, OctC4) and β-hairpin peptides (arenicin-3, tachyplesin) generally retain activity similar to that of the parent antibiotic, both as the azide, and with the more disruptive fluorophore substitutent. OctC4 is a Pmx analogue discovered in the 1970s. Despite its structural similarity to PmxB, OctC4 retains activity against Pmx-resistant strains of bacteria, and is much less prone to inducing resistance.54 For both PmxB and OctC4, the azido- and fluorophore-antibiotics retained antimicrobial potency (Table 1). Importantly, the polymyxin and octapeptin analogues retained their parent antibiotic selectivity against Pmx-sensitive and –resistant strains, providing an opportunity to apply the probes to mode of action studies to investigate why the closely related antibiotics vary in activity. In contrast, antibiotic probes derived from antibiotics acting on intracellular targets (linezolid,52 trimethoprim (TMP),53 metronidazole and roxithromycin) tended to have reduced activity once the fluorophore was added to the azide, with roxithromycin the exception (Table 1). In the case of TMP, the TMP-based fluorescent probes (TMP-F*) had little antibacterial activity, yet still stained inside G+ve and G-ve bacteria (data not shown). Inhibition assays of the


dihydrofolate reductase enzymatic target showed similar potency as TMP. MIC testing against E. coli mutant strains with a deficient efflux pump (TolC) or permeable membrane (DC2) demonstrated that the loss of MIC activity was due to excessive efflux. Significantly higher intracellular fluorescence was observed in both wild type and ΔtolC E. coli when TMP-F* was added to cells pre-treated with an efflux pump inhibitor, CCCP, showing the utility of this probe for evaluating efflux pump inhibition.53

Table 1. Minimum Inhibitory Concentrations of azide functionalised antibiotics.

Parent Antibiotic Bacteria MIC (µg/mL)

Parent Antibiotic

Antibiotic -N3

X = Tz-NBD X = Tz-DMACA vancomycin MRSA a 1 1 0.25 0.5

linezolid MRSA a 2 16 16 >64

trimethoprim

MRSA a E. coli b TolC c DC2 d

1 1

0.25 0.5

2 4

0.125 2

32 >64

2 >64

>64 >84

8 >64

metronidazole Clostridium difficile e 0.5 1 8 N/A

polymyxin B (PmxB)

E. coli b Pseudomonas aeruginosa f

PmxR P. aeruginosa g

0.125 0.25 >64

0.125 0.25 >64

0.25 0.5 >64

1 2

>64

octapeptin C4 (OctC4)

E. coli b P. aerug f

PmxR P. aeruginosa g

4 2 1

4 2 2

2 1 1

4 2 4

arenicin-3 E. coli b 0.5 2 8 N/A tachyplesin E. coli b 0.03 0.125 0.25 N/A

roxithromycin MRSA a 0.25 1 1 8 a methicillin-resistant Staphylococcus aureus ATCC 43300, b ATCC 25922, c E. coli efflux pump mutant, d E. coli

membrane mutant, e ATCC BAA-1382, f ATCC 27853, g polymyxin-res P. aeruginosa FADDI-PA070

3.2 Fluorescence Microscopy Super-resolution structured illumination microscopy (SR-SIM) studies of vancomycin probes demonstrated they bound to the cell wall of G+ve bacteria with a high concentration at the dividing septum, the site of new peptidoglycan synthesis, as reported for previous vancomycin-derived probes (Figure 3). The advantage of the ‘click’ chemistry approach is highlighted by the ease at generating multiple colour probes (Figure 3A-C) As expected, the vancomycin-NBD probe did not normally stain G-ve bacteria such as E. coli since the outer-membrane protects the peptidoglycan layer (Figure 3D), but a membrane-impaired lpxC mutant showed substantial internal labelling (Figure 3E). The polymyxin (Figure 3F) and octapeptin (Figure 3G) probes localised in the membrane of E. coli, the site of their Lipid A target, as expected. In contrast, probes derived from antibiotics expected to act on internal targets, such as linezolid-NBD, are found inside the bacteria (Figure 3H), speculatively due to binding to the linezolid 50S ribosome target. SR-SIM provides enough resolution to distinguish the vancomycin-DMACA labeled peptidoglycan layer (blue) sitting outside the membrane (FM4-64X-dye, red) (Figure 3I), with strong internal signal from linezolid-NBD (green).

3.3 Microfluidic Single Cell Tracking Preliminary testing of the uptake of the roxithromycin-NBD probe in individual cells of E. coli using microfluidic delivery of the probe to cells immobilized within channels of a chip55 showed substantial heterogeneity in timing of probe uptake (Figure 4), in contrast with S. aureus (data not shown) where uptake was much more uniform. Monitoring of single cells over time will allow for assessment and analysis of resistance vs persistence phenotypes.


Figure 3. A-C. SR-SIM vancomycin-fluorophore labelling of S. aureus ATCC 25923 showing strong septum localisation (bacteria ~0.6 µm), including 3D Image reconstruction SIM of S. aureus with Van-NBD probe. D Wild-type or E mutant lpxC E. coli with Van-NBD and FM4-64X (red, membrane dye) showing outer membrane penetration to peptidoglycan limited to the division site in

normal bacteria but substantially increased with damaged OM. F-G. Staining of E. coli ATCC 25922 by polymyxin-NBD or octapeptin-DMACA probes showing membrane localisation. H. Linezolid-NBD probe showing internalisation in S. aureus ATCC

25923, possibly at ribosome target. I. Co-staining of S. aureus ATCC 25923 with green Linezolid-NBD, blue Van-DMACA and red FM4-64X membrane dye, with cross section showing resolution of peptidoglycan and membrane labelling.

Figure 4. Single cell uptake of roxithromycin-NBD probe by E. coli after 140 min of exposure in microfluidics channel: yellow box =

channels with no bacteria, magenta box = individual cells with no uptake surrounded by cells with substantial fluorescence.


3.4 PET Imaging Probes In order to extend the functionalised antibiotic approach to probes that could be applied to whole-body imaging, we have functionalised the radioisotope metal chelator DOTA, (1,4,7,10-tetra-azacyclododecanetetraacetic acid) with an alkyne substituent and coupled this with the vancomycin-azide intermediate. The adduct retained antimicrobial activity, and preliminary labeling studies with the PET isotope copper-64 showed quantitatively labeling at room temperature. However treatment of the reaction solution with excess of EDTA revealed that only 40% of the copper-64 bound strongly to the DOTA chelate at neutral pH, with 60% able to be displaced by EDTA. Vancomycin is known to bind copper;56 so presumably the weakly bound component results from vancomycin complexation. The kinetics of copper complexation to both vancomycin and the chelator requiring further optimization of reaction conditions to preferentially bind the copper-64 within the DOTA complex: initial experiments show that pH adjustment leads to over 90% ‘strong’ binding not displaced by EDTA.

4. CONCLUSIONS We have developed a platform based on functionalised antibiotics that has delivered a pipeline of mechanistic-specific fluorescent probes. These are being employed for antibiotic mode of action and bacterial resistance studies, and for the development of assays capable of rapidly characterizing bacterial resistance. We are keen to share these probes with collaborators to utilize their full potential.

We have also initiated studies to convert these probes into diagnostics suitable for whole body imaging of infections. To date, diagnostic approaches for imaging infections have generally relied on non-specific probes that are not effective at identifying infections, and are particularly poor at discriminating between infection and inflammation. The key innovations in our approach are that: 1) we have identified small molecule scaffolds that are very selective at binding to bacteria over other cells, and 2) we have modified these intermediates to readily attach different imaging moieties, allowing for rapid optimisation.

ACKNOWLEDGEMENTS

MRLS is supported by an Australian Postgraduate Award (APA) PhD scholarship. WP was supported by a UQ International Scholarship (UQI) and IMB Postgraduate Award (IMBPA). MATB is supported in part by Wellcome Trust Strategic Grant WT1104797/Z/14/Z and NHMRC Development grant APP1113719. M.A.C. is a NHMRC principle research fellow (APP1059354) and also holds a fractional professorial research fellow appointment at the University of Queensland, with his remaining time as CEO of Inflazome Ltd, a company developing drugs to address clinical unmet needs in inflammatory disease. SR-SIM microscopy at the Queensland Brain Institute is supported by ARC LIEF grant LE130100078. Microscopy was also performed at the Australian Cancer Research Foundation (ACRF)/Institute for Molecular Bioscience Cancer Biology Imaging Facility, which was established with the support of the ACRF. The mutant tolC, lpxC and DC2 E. coli strains MB4902, MB5746, MB5747 were generously supplied by Merck Sharp & Dohme (Kenilworth, NJ).

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