Starving bacteria of iron: a potential strategy to disperse bacterial biofilms

Abstract

Halogenated phenazines (HPs) are potent antimicrobial agents. A newly developed halogenated phenazine, HP-29, displays remarkable minimum inhibitory concentration (MIC) of 0.08 μM against methicillin-resistant Staphylococcus aureus, MRSA. HP-29 eradicates preformed biofilm via iron starvation, is non-toxic to mammalian cell lines and is efficacious in wound infection models.

Introduction:

Antimicrobial resistance is a major health issue, which requires good antibiotic stewardship as well as the development of novel agents against emerging bacterial threats. An influential report, chaired by Lord O’Neill, predicted that by 2050 antimicrobial resistance infections could kill 10 million people annually if it is not combatted soon.¹ While others may contend with the high death estimate in Lord O’Neill’s report,² even going by current statistics death from antimicrobial resistant infections remain high. In 2019, the CDC reported that more than 2.8 million antibiotic-resistant infections occur in the U.S. each year, resulting in more than 35,000 deaths.³ Of these drug-resistant pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) have been recognized as high-priority pathogens by the WHO as well as serious threats by the CDC. For instance, MRSA causes 323,700 infections and 10,600 deaths annually, while VRE results in 54,500 infections and 5,400 deaths yearly.³

Most MRSA and VRE infections are exacerbated by biofilm formation, which contribute to the infections becoming chronic. Biofilm is a cluster of bacterial pathogens that anchors to either biological or non-biological surfaces. Biofilm bacteria are estimated to be 10-1,000 times more resistant to antibiotics than planktonic bacteria.⁴ Current FDA-approved antibiotics do not eradicate preformed MRSA or VRE biofilms.⁵ New antibiotic chemotypes that eradicate MRSA and VRE biofilms are therefore needed to fight these pathogens.

The Huigens III group previously reported that halogenated phenazines have biofilm eradication activities.^6,7 In their previous works, the phenazines were synthesized via phenylenediamine condensations or Wohl-Aue reaction. The phenylenediamine reaction suffers from low regioselectivity, whiles the Wohl-Aue reaction suffers from low reaction yields and many side products. These synthesis bottlenecks have hampered access to phenazines analogs substituted at position-3 (see Figure 1, compound 3 for numbering), to complete structure-activity-relationship studies. In this work published in the Journal of Medicinal Chemistry, the Huigens III group reports that halogenated phenazine (HP) analogs, which are substituted at position-3 with chloro or other groups, can be readily synthesized from anilines and 2-nitro-5-chloroanisole via N-aryl-2-nitrosoaniline intermediate. The N-aryl-2-nitrosoaniline intermediate facilely cyclizes to phenazines using N,O-bis(trimethylsilyl)acetamide in DMF solvent, allowing for the synthesis of 3-chloro substituted phenazine analogs.⁸ The chloro group can act as a synthetic handle, allowing for S_NAr reactions with thiol nucleophiles. Using this streamlined synthesis, over 20 HP analogs were synthesized and screened for activity against several bacterial strains like MRSA, VRE, and methicillin-resistant Staphylococcus epidermidis (MRSE). It was discovered that the nature and pattern of phenazine substitution affected both bacterial growth inhibition and biofilm eradication properties of the compounds. As illustrative examples, compounds HP-45 and HP-29 differ only at position-3 substitution (SEt versus Cl) but display remarkably different minimum biofilm eradication concentration (MBEC) of 200 μM and 2.35 μM respectively against MRSA-1707. Compound 3, which lacks substitution at both position-3 and -6 has MBEC of 100 μM against MRSA-1707. All three compounds have good growth inhibitory properties with minimum inhibitory concentration (MIC) of 1.56 μM (Cpd. 3), 0.08 μM (HP-29), 0.3 μM (HP-45) against MRSA-1707. Under similar experimental conditions, the MICs for vancomycin, daptomycin and linezolid (standard drugs for MRSA) were 0.39 μM, 3.13 μM and 12.5 μM respectively.

Figure 1.

Halogenated Phenazines were synthesized from the N-aryl-2-nitrosoaniline intermediate. a) Cyclization with N,O-bis(trimethylsilyl)acetamide, followed by S_NAr reactions with thiol nucleophiles and bromination with NBS; b) Cyclization with N,O-bis(trimethylsilyl)acetamide, followed by demethylation with BBr₃ and bromination with NBS.

In addition to growth inhibition and biofilm eradication properties, Huigens III and co-workers showed that the halogenated phenazines eradicate biofilms by inducing iron starvation (Figure 2). The group used RT-qPCR to determine that HP-29 induced rapid up-regulation of genes involved in iron uptake (isdB, sfaA, sbnC, and MW0695). Similarly, HP-29 was found to directly bind iron (II) via UV-vis spectroscopy, which probably explains the RT-qPCR results.

Figure 2.

HP-29 causes iron starvation in bacteria, leading to biofilm eradication. HP-29 was found to be highly efficacious in MRSA and VRE wound infection models.

To demonstrate the translational potential of the new 3-substituted halogenated phenazines, the group determined that all HP analogs were non-toxic to mammalian cells at concentrations greater than 200 μM. Additionally, the HPs also had little to no hemolytic activity against red blood cells at concentrations of 200 μM. HP-29 was assessed for in vivo efficacy in MRSA and VRE wound infections. HP-29 treated mice had a 0.82-log₁₀ reduction in CFU per lesion of MRSA when compared to vehicle. Likewise, HP-29 treated mice had a 1.73-log₁₀ reduction in CFU per lesion of VRE when compared to vehicle.

In summary, this study presented by Huigens III et al. features a new class of antibiotics to treat drug-resistant Gram-positive bacteria. In particular, HP-29 showed highly potent activity against MRSA, VRE, and MRSE with MICs ranging from 0.08 μM to 0.30 μM. Additionally, HP-29 was found to be non-toxic against mammalian cell lines and does not lyse red blood cells. HP-29 also potently eradicates preformed biofilms via direct binding to iron(II), resulting in iron starvation. Furthermore, HP-29 was also efficacious in both MRSA and VRE mouse wound infection models. Overall, the compounds reported in this study add to the list of potential antimicrobial agents we could use during the impending doom of multi-drug resistant bacterial infections era. However, for this class of compounds to be successful as agents for systemic infection, a few potential concerns need to be addressed, such as solubility concern and the propensity of drugs containing heavy halogens such as bromine and iodine to show phototoxicity. With regards to the mode of action of the compounds, the presented evidence points to iron starvation as a probable contributing factor accounting for the biofilm eradication. However iron binding alone does not explain the differential biofilm eradicating properties of the phenazine analogs/other iron binding compounds. Future experiments that investigate cellular target binding could help provide more insights into the mode of biofilm eradication by these compounds.