Identification of 6,8-ditrifluoromethyl halogenated phenazine as a potent bacterial biofilm-eradicating agent

Abstract

Bacterial biofilms are surface-attached communities consisting of non-replicating persister cells encased within an extracellular matrix of biomolecules. Unlike bacteria that have acquired resistance to antibiotics, persister cells enable biofilms to demonstrate innate tolerance toward all classes of conventional antibiotic therapies. It is estimated that 50-80% of bacterial infections are biofilm associated, which is considered the underlying cause of chronic and recurring infections. Herein, we report a modular three-step synthetic route to new halogenated phenazine (HP) analogues from diverse aniline and nitroarene building blocks. The HPs were evaluated for antibacterial and biofilm-killing properties against a panel of lab strains and multidrug-resistant clinical isolates. Several HPs demonstrated potent antibacterial (MIC ≤ 0.39 μM) and biofilm-eradicating activities (MBEC < 10 μM) with 6,8-ditrifluoromethyl-HP 15 demonstrated remarkable biofilm-killing potencies (MBEC = 0.15-1.17 μM) against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus clinical isolates. Confocal microscopy showed HP 15 induced significant losses in the polysaccharide matrix in MRSA biofilms. In addition, HP 15 showed increased antibacterial activities against dormant Mycobacterium tuberculosis (Mtb, MIC = 1.35 μM) when compared to replicating Mtb (MIC = 3.69 μM). Overall, this new modular route has enabled rapid access to an interesting series of potent halogenated phenazine analogues to explore their unique antibacterial and biofilm-killing properties.

Fig. 1. Individual, free-floating planktonic bacteria coordinate the simultaneous attachment to a surface where subsequent development leads to a mature biofilm community consisting of enriched populations of non-replicating (metabolically-dormant) persister cells. Established biofilms will disperse planktonic cells back into the surrounding environment and are credited as the underlying cause of chronic and recurring bacterial infection.

Fig. 2. Halogenated phenazine (HP) analogues that have demonstrated potent biofilm eradication activities and/or have been used as probes to explore mechanistic insights.

Scheme 1. Modular synthesis of halogenated phenazines from diverse nitroarene and aniline materials. Notes. (a) Demethylation or bromination yields from previous studies. (b) Previously reported halogenated phenazines.

Fig. 3. (A) Chemical structures and focused antibacterial profiles (against MRSA 1707 & MRSE 35984) for potent halogenated phenazines investigated during these studies. (B) Images of representative MIC assays against MRSA 1707 and MRSE 35984. (C) UV-vis experiments to show select HPs directly binding iron(ii).

Fig. 4. (A) Select halogenated phenazine biofilm eradication activities. (B) Representative MBEC images of halogenated phenazines evaluated in Calgary Biofilm Device (CBD) assays. (C) Biofilm eradication profile for HP 15. Note: *Compounds were tested from 0.2 μM to 200 μM (10 mM DMSO stock solution). **Compounds were tested from 0.1 μM to 100 μM (5 mM DMSO stock solution). ***Compounds were tested from 2 μM to 2000 μM (100 mM DMSO stock solution).

Fig. 5. Confocal microscopy images of MRSA biofilms untreated or treated with select HP analogues. The figures show the 3-D rendering of confocal z-stack imaging of biofilms grown with GFP expressing S. aureus and stained with Texas Red conjugated concanavalin A. The top three images show MRSA biofilms with no compound (DMSO Control), the middle and bottom images are those treated with 25 μM of HP-1 and 6,8-ditrifluoromethyl-HP 15, respectively. All imaging was done with n = 3 replicates with representative images shown.