Halogenated Phenazines that Potently Eradicate Biofilms, MRSA Persister Cells in Non‐Biofilm Cultures, and Mycobacterium tuberculosis

Abstract

Conventional antibiotics are ineffective against non-replicating bacteria (for example, bacteria within biofilms). We report a series of halogenated phenazines (HP), inspired by marine antibiotic 1, that targets persistent bacteria. HP 14 demonstrated the most potent biofilm eradication activities to date against MRSA, MRSE, and VRE biofilms (MBEC = 0.2-12.5 μM), as well as the effective killing of MRSA persister cells in non-biofilm cultures. Frontline MRSA treatments, vancomycin and daptomycin, were unable to eradicate MRSA biofilms or non-biofilm persisters alongside 14. HP 13 displayed potent antibacterial activity against slow-growing M. tuberculosis (MIC = 3.13 μM), the leading cause of death by bacterial infection around the world. HP analogues effectively target persistent bacteria through a mechanism that is non-toxic to mammalian cells and could have a significant impact on treatments for chronic bacterial infections.

A Highly Potent Class of Halogenated Phenazine Antibacterial and Biofilm-Eradicating Agents Accessed Through a Modular Wohl-Aue Synthesis

“…From our previous studies, we have found that biofilm eradication activities correlate well to antibacterial activities for HP small molecules 24, 25 . With this in mind, we advanced a focused set of Wohl-Aue derived HP analogues to biofilm eradication assays using Calgary Biofilm Devices (CBD) 42, 43 containing specialized 96-well plates with a lid containing 96 pegs anchored to the lid to provide a surface for biofilms to form and be treated (one peg per microtiter well).…”

“…With active HPs targeting both rapidly-dividing planktonic and persistent biofilm cells of Gram-positive pathogens, we felt that HPs could demonstrate antibacterial activities against the persistent pathogen, MtB. We previously reported HPs with good anti-TB activity 24, 25 and, during the course of these studies, we evaluated Wohl-Aue derived HPs for antibacterial activity against MtB H37Ra. We tested a smaller panel of HPs ( 9 , 17 , 18 , 21–23 ; Table 1) that demonstrated potent antibacterial activities against MRSA, MRSE, and VRE.…”

“…We previously demonstrated one HP analogue slowly kills MRSA persister cells in kinetic kill experiments of stationary cultures 24 , which are known to contain higher populations of non-replicating persister cells 46, 47 . We were curious to see if the biofilm eradication activities of HPs would show a more potent effect if MRSA biofilms were pulse treated with active HPs (i.e., two subsequent 24 hour compound treatment phases).…”

“…Our pursuits have been met with challenges regarding the chemical synthesis of the HP scaffold. Our previous work has enabled the exploration of 7,8-disubstituted HPs along with 2,4-mixed halogenations HPs with the use of o -phenylenediamines building blocks 24, 25 ; however, the availability of these building blocks does not enable the level of diversity necessary for our campaign. Our goal for this work is to develop a rapid and convergent synthesis of HP small molecules that enable us to further explore the 6-, 7-, 8- and 9-positions of the HP scaffold through the use of diverse aniline building blocks.…”

Phage Abp1 Rescues Human Cells and Mice from Infection by Pan-Drug Resistant Acinetobacter Baumannii

“…LDH release from HeLa and THP-1 cells was determined using a CytoTox96 cytotoxicity assay (Promega G1780) [12]. HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium and THP-1 cells in Roswell Park Memorial Institute 1640 Medium, both supplemented with fetal bovine serum (10%) and penicillin- streptomycin (1%), and incubated in 5% CO 2 at 37°C.…”