key: cord-0430722-6z850760
authors: Murphy, Ronan A.; Coates, Matthew; Thrane, Sophia; Sabnis, Akshay; Harrison, James; Schelenz, Silke; Edwards, Andrew M.; Vorup-Jensen, Thomas; Davies, Jane C.
title: Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin Against Cystic Fibrosis Pseudomonas aeruginosa
date: 2022-02-03
journal: bioRxiv
DOI: 10.1101/2022.02.03.478806
sha: 20848b5cedbb63986090235d495ffa56704856b5
doc_id: 430722
cord_uid: 6z850760

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonise and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate anti-pseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers; potentiating the activities of antibiotics when given in combination restoring and/or enhancing antibiotic efficacy. Growth, viability, minimum inhibitory concentration (MIC)-determination and synergy analysis showed that GA improved the efficacy of TOB against reference strains of P. aeruginosa, reducing TOB MICs and synergising with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the concentration of TOB required to inhibit 50% (MIC50) of viable cells (from 1.69 [95%CI 0.26-8.97] to 0.62 [95%CI 0.15-3.94] mg/L, p=0.002) and inhibit 90% (MIC90) (from 7.00 [95%CI 1.18-26.50] to 2.20 [95%CI 0.99-15.03] mg/L, p=0.001) compared with TOB-only. Investigating mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarisation of cytoplasmic membranes and permeabilisation of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three.

antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, 23 allow P. aeruginosa to colonise and persist in spite of antibiotic treatment. As well as the specific 24 difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium found ubiquitously 42 in the environment and frequently associated with opportunistic infections (burns, wounds, eye-43 infections). P. aeruginosa is the most common infecting bacteria in the lungs of people with cystic 44 fibrosis (CF) (1). Cystic fibrosis is a genetic, life-limiting disorder and, while a multi-system illness, 45 lung disease causes the majority of morbidity and mortality in people with CF; impaired 46 mucociliary clearance leads to chronic bacterial infection, significant inflammation and 47 bronchiectasis resulting, ultimately, in respiratory failure (2-4). Forty-one percent of adult CF 48 patients (>16 years) in the UK are chronically infected with P. aeruginosa, with a peak of 54% in 49 the 36-39 age cohort (1). P. aeruginosa is of particular concern due its ability to evade antibiotic 50 treatments (via both innate and acquired mechanisms) leading to its designation as an ESKAPE 51 pathogen by the World Health Organisation (5-7). 52 The aminoglycoside, tobramycin is one of the most commonly used anti-pseudomonal 53 antibiotics in cystic fibrosis. Administration is either intravenous (IV) or inhaled directly to the 54 airway (1). Limitations of the IV route include a narrow therapeutic index requiring concentration 55 monitoring to avoid oto-and nephrotoxicity. The inhaled route allows high local levels with less 56 systemic exposure, but once infection is chronic, drug efficacy is modest and eradication rare, a 57 limitation shared with other agents at this disease stage. In part, this may relate to heterogeneity 58 of deposition within partially obstructed airways, giving rise to subtherapeutic drug 59 concentrations. Few new treatments are being developed for chronic P. aeruginosa, and the 60 4 growing adult population means this unmet need will persist into the era of novel modulator 61 drugs targeting the underlying cellular defect in CF (8,9). 62 A potential solution to the need for novel P. aeruginosa therapy, among the dearth of 63 new antibacterial treatments, is the deployment of antimicrobial peptides (AMPs), small 64 biological molecules, usually consisting of 10-50 amino acid residues, which form part of innate 65 immune systems produced widely across all kingdoms of life (10) (11) (12) . They frequently function 66 via interactions with the bacterial membrane which lead to cell wall weakening, thinning and/or 67 permeabilisation and rapid cell death (10, (13) (14) (15) . The Gram-negative membrane consists of an 68 outer membrane, containing outwards protruding lipopolysaccharide, and a cytoplasmic 69 membrane with a thin peptidoglycan layer between. AMPs are of interest not only because of 70 their own potential antimicrobial activity but also their possible use as antibiotic adjuvants; given 71 in combination with antibiotics, they may reduce concentrations of the latter required for 72 antimicrobial activity (16). This property, often referred to as 'antibiotic resistance breaking', has 73 been reported against P. aeruginosa: the human-derived AMP LL-37 and its derivatives with 74 azithromycin, vancomycin (17,18), novel synthetic peptides with tobramycin and colistin (19-21). 75 However, despite promising results, many AMPs have significant barriers to clinical use, including 76 concerns around cytotoxic effects (13,22,23). 77 Glatiramer acetate (GA) is an immunomodulator drug currently in clinical use in the 78 treatment of multiple sclerosis (24) (25) (26) . It is produced by the random polymerisation of four 79 amino acids (L-glutamate, L-lysine, L-alanine, and L-tyrosine residues) and therefore structurally 80 resembles AMPs. We have shown previously that GA has moderate antimicrobial activity 81 comparable to LL-37 against P. aeruginosa -both reference strains and clinical strains from 82 5 people with CF, optimal activity occurring at 50mg/L (15,27). GA has other properties in common 83 with naturally occurring antibiotic resistance breaking AMPs being cationic, <150 amino acid 84 residues long and exerting antimicrobial effects over an extremely short time course to a level 85 similar to that of the AMP LL-37 (26,28). 86 Investigating existing drugs which are not conventional antibiotics for their potential use 87 as antimicrobials, known as 'repurposing', is a growing area of interest and research (29,30). As 88 well as addressing the paucity of new antibiotics being developed, repurposing of existing drugs 89 has several other advantages: approved drugs have historical safety data and repurposing may 90 minimise time and costs associated with deployment to the clinic due the availability of previous 91 trials and research (30). 92 As with other members of the aminoglycoside family, tobramycin targets the bacterial 93 protein synthesis machinery and thus requires uptake into the bacterial cytoplasm for activity 94 (31). Given its widespread use in CF, preserving, restoring and/or enhancing the efficacy of 95 tobramycin is of particular interest and importance. As it has also been previously subject to 96 'breaking' by antimicrobial peptides, we examined tobramycin in combination with GA, to assess 97 the peptide as a potential antibiotic resistance breaker in cystic fibrosis strains of P. aeruginosa. 98 We quantified the impact of GA on bacterial survival at a range of tobramycin concentrations and 99 assessed its direct effects on outer and cytoplasmic membranes to elucidate mechanisms of 100 action. (Figure 2 ). The impact on PAK was 241 visually similar, but did not reach statistical significance (Figure 2 ). 242 We further analysed these CFU data to assess whether any impact of GA on TOB was 243 additive or synergistic , the latter defined as the combined effect of the two agents being greater 244 than would be predicted by the individual effects seen for each (EOBs > EExp). For PAO1 the 245 combination of GA with both 0.25mg/L and 0.5mg/L TOB was synergistic; for PA14, synergy was 246 observed for GA with 1mg/L TOB; for PAK GA with 2mg/L TOB was synergistic (Figure 2 ). Glatiramer acetate disrupts the outer bacterial membrane of P. aeruginosa 292 We next wished to explore the mechanism by which GA was potentiating the activity of 293 TOB. The fluorescent probe, NPN, was used to measure disruption of the outer membrane of P. We hypothesised that the peptide drug, glatiramer acetate, would be an antibiotic 337 resistance breaker in P. aeruginosa, when combined with the aminoglycoside, tobramycin. Here, 338 we provide data to support this hypothesis and demonstrate the drug's mechanism of action, 

UK Cystic Fibrosis Registry Annual 458 data report

Origins of Cystic Fibrosis Lung Disease

Cystic fibrosis

Cystic fibrosis

Federal funding for the study of antimicrobial resistance in nosocomial 464 pathogens: No ESKAPE

Clinical relevance of the ESKAPE pathogens

World Health 468 Organization. World Health Organisation

New anti-pseudomonal agents for cystic fibrosis-still needed in the 470 era of small molecule CFTR modulators

Current and 472 future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis

Antimicrobial peptides: Has their time arrived? Future Microbiol. 475 2015/06/30

The antimicrobial peptides and their 477 potential clinical applications

DRAMP 3.0: an enhanced comprehensive data repository 480 of antimicrobial peptides

Antimicrobial peptides

Antimicrobial peptides of multicellular organisms

The Immunomodulatory Drug Glatiramer Acetate is Also an Effective Antimicrobial Agent 486 that Kills Gram-negative Bacteria

Antibiotic Adjuvants: Rescuing Antibiotics from Resistance (Trends 488 in Microbiology

Azithromycin Synergizes 490 with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity 491

Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens

Synthetic Peptide Improves Therapeutic Potential of Vancomycin Against Pseudomonas 495 aeruginosa

Assessing the synergistic effect of the 497 enhancer compound HT61 on Tobramycin activity in a murine model of pulmonary 498 infection

The small 501 quinolone derived compound HT61 enhances the effect of tobramycin against 502

Pseudomonas aeruginosa in vitro and in vivo

Synergistic activity of a short lipidated antimicrobial peptide (lipoAMP) and colistin or 506 tobramycin against Pseudomonas aeruginosa from cystic fibrosis patients

Analysis of the cytotoxicity of synthetic 509 antimicrobial peptides on mouse leucocytes: Implications for systemic use

LL-37-derived antimicrobial peptide in an animal model of biofilm Pseudomonas sinusitis

Multiple sclerosis: Trial of a 515 synthetic polypeptide

Specific inhibition of the T-cell response to 517 myelin basic protein by the synthetic copolymer Cop 1

The 520 random co-polymer glatiramer acetate rapidly kills primary human leukocytes through 521 sialic-acid-dependent cell membrane damage

Inhaled nebulized glatiramer acetate against Gram-negative bacteria is not associated 525 with adverse pulmonary reactions in healthy, young adult female pigs

Antimicrobial peptides: Application informed by evolution

The global preclinical antibacterial 530 pipeline

Drug repurposing for the treatment of 532 bacterial and fungal infections

Binding of aminoglycosidic antibiotics to the

A-site model and 30S ribosomal subunit: Poisson-Boltzmann model, 535 thermal denaturation, and fluorescence studies

The estimation of the bactericidal power of the blood

Colistin kills 539 bacteria by targeting lipopolysaccharide in the cytoplasmic membrane

The Immunomodulatory Drug Glatiramer Acetate is Also an Effective Antimicrobial Agent 543 that Kills Gram-negative Bacteria

Human host 545 defense peptide LL-37 Stimulates virulence factor production and adaptive resistance in 546 Pseudomonas aeruginosa

Mechanism of 548 Action of a Membrane-Active Quinoline-Based Antimicrobial on Natural and Model 549 Bacterial Membranes

A new bliss 551 independence model to analyze drug combination data

Resistance mechanisms in Pseudomonas aeruginosa and other

Permeability of Pseudomonas aeruginosa outer membrane to 556 hydrophilic solutes

Role of Membrane Permeability in Determining Antibiotic Resistance in 558 Pseudomonas aeruginosa

Aminoglycoside-resistance mechanisms for cystic fibrosis Pseudomonas aeruginosa 561 isolates are unchanged by long-term, intermittent, inhaled tobramycin treatment

564 Characterization of a Pseudomonas aeruginosa efflux pump contributing to 565 aminoglycoside impermeability

Contribution of the MexXY multidrug transporter to 567 aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. Antimicrob 568 Agents Chemother

Multiple antibiotic resistance in Pseudomonas 570 aeruginosa: Evidence for involvement of an efflux operon

Aminoglycoside resistance in Pseudomonas aeruginosa

Binding of polycationic antibiotics and 575 polyamines to lipopolysaccharides of Pseudomonas aeruginosa

PhoQ 578 mutations promote lipid A modification and polymyxin resistance of Pseudomonas 579 aeruginosa found in colistin-treated cystic fibrosis patients

4-Amino-4-582 deoxy-L-arabinose in LPS of enterobacterial R-mutants and its possible role for their 583 polymyxin reactivity

PmrAB, a Two-Component Regulatory System of 585

Pseudomonas aeruginosa that Modulates Resistance to Cationic Antimicrobial Peptides 586 and Addition of Aminoarabinose to Lipid A

Electrostatic modification of the lipopolysaccharide layer: 588 Competing effects of divalent cations and polycationic or polyanionic molecules

Membrane Reveals Cation Displacement and Increasing Membrane Curvature in 592 Susceptible but Not in Resistant Lipopolysaccharide Chemotypes

Release of lipopolysaccharide by EDTA treatment of E. coli

A Nonspecific Increase in Permeability in Escherichia Coli Produced By Edta

Studies on the permeability change produced in coliform bacteria by 599 ethylenediaminetetraacetate

Role of divalent cations in the action of polymyxin B and EDTA on 601 Pseudomonas aeruginosa

Loss of sensitivity to EDTA by Pseudomonas aeruginosa grown 603 under conditions of Mg limitation

Antibiotic 605 prescribing in patients with COVID-19: rapid review and meta-analysis

Inappropriate antibiotic use in the COVID-19 era: Factors 609 associated with inappropriate prescribing and secondary complications

Characterising 612 burden of treatment in cystic fibrosis to identify priority areas for clinical trials

Prevalence and 615 characteristics of chronic kidney disease among Danish adults with cystic fibrosis

Prevalence of hearing and vestibular loss in 618 cystic fibrosis patients exposed to aminoglycosides

A comparison of peak sputum tobramycin concentration in patients with cystic 622 fibrosis using jet and ultrasonic nebulizer systems

Pharmacokinetics and 624 bioavailability of aerosolized tobramycin in cystic fibrosis

Semilog plots of overnight growth of P. aeruginosa PAO1 (natural logs of OD600, 644 median and 95% confidence intervals) at increasing concentrations of TOB

Combination effect of GA/TOB was noted at 0.5mg/L TOB where the final OD600 of the growth 646 curves were significantly lower for GA/TOB than TOB (p<0.05). B. Heat Map of Areas Under the 647 Curve of PAO1, PA14 and PAK OD600 overnight growth curves at all TOB concentrations tested 648 (median of at least three biological replicates). Darkness of red indicates lower AUCs and less 649 bacterial growth. For PAO1 and PAK, GA/TOB

Colony forming units of strains PAO1, PA14 and PAK after overnight exposure to TOB 653 and GA/TOB at increasing TOB concentrations (median with 95% confidence intervals). * 654 indicates concentrations for which GA/TOB resulted in significantly fewer viable bacteria that 655

TOB (p<0.05). † indicates the TOB concentrations where GA/TOB displayed synergy (EOBs > EExp)

Shaded areas indicate results which where CFU/mL were significantly lower than untreated P

aeruginosa (data not shown on graphs: stable CFU/ml value as seen with lowest concentrations 658 of TOB) for a given strain (p<0.05). For each strain, the highest GA/TOB concentration tested 659 eliminated viable bacteria, whereas the same TOB-only treatment did not

Figure 3. MIC50 and MIC90 values of TOB for strains PAO1, PA14 and PAK in the absence and 662 presence of GA

MIC50 and MIC90 results for TOB for 11 clinical P. aeruginosa strains from cystic fibrosis 667 in the absence and presence of GA. Values were interpolated from non-linear fit of inhibition 668 curves of CFU/mL results from at least 3 biological replicates for each strain. Across the 11 strains 669 tested both MIC50 and MIC90 values of TOB were significantly reduced by co-administration of GA 670

Comparison of uptake of NPN over 15mins by the outer membranes of strains PAO1, 673 PA14 and PAK resulting from exposure to 50mg/L GA, 2mg/L CST, 4mg/L TOB and

GA caused a significant increase in outer 676 membrane disruption for all three reference strains

Effect of 50mg/L GA on the A

Each point represents a strain and is the 680 median of at least three independent experiments. Line at median of all clinical strains tested

GA caused significant disruption of bacterial outer membrane, significant depolarisation of the

cytoplasmic membrane and significant permeabilisation of the cell wall

Comparison of release of DiSC3(5) over 15mins by the cytoplasmic membranes of strains 686

PAO1, PA14 and PAK resulting from exposure to 50mg/L GA, 2mg/L CST, 4mg/L TOB and 16mg/L

GA caused a significant 689 depolarisation of cytoplasmic membranes for all three reference strains

Comparison Areas Under the Curve of fluorescence of propidium iodide over 1hr of 692 strains PAO1, PA14 and PAK resulting from exposure to 50mg/L GA, 2mg/L CST

GA caused a significant increase in 695 bacterial cell permeabilisation of all three reference strains