Development of Antibiotic Adjuvants to Avert Resistance Conferred by Cfr, a Radical S-Adenosylmethionine Methylase

Project Summary/Abstract
Prevalent multidrug-resistant bacteria remain a significant threat to human health. About 1 million people,
including 214,000 newborn babies, die annually from such infections. Most antibiotics typically target essential
cellular components, enacting a high selective pressure that forces bacteria to develop resistance. This factor,
among others, led to dire statistical projections citing 10 million annual deaths by 2050, prompting a call-to-
action directed at tackling resistance through novel strategies. The most prominent bacterial resistance
mechanisms include genetically encoded efflux pumps, structural modification of antibiotics, or modification of
their molecular targets. Many target modification events follow well-established mechanisms catalyzed by
enzymes. For example, the documented loss of activity by multiple classes of antibiotics that target protein
synthesis is attributed to modification of ribosomal RNA (rRNA) through methylation by S-adenosylmethionine
(SAM)-dependent methylases. The Chloramphenicol-florfenicol resistance (Cfr) protein is a radical SAM-
dependent enzyme that catalyzes the methylation of adenosine 2503 (A2503) of 23S rRNA in the large subunit
of bacterial ribosomes, conferring resistance to several classes antibiotics that inhibit translation. The gene
encoding Cfr is found on naturally occurring plasmids across multiple pathogenic bacteria including
Staphylococcus, Clostridium, Enterococcus, Listeria, and Bacillus species. This proposal focuses on developing
compounds that inhibit the activity of Cfr, thereby preventing bacteria from acquiring resistance through rRNA
methylation by this enzyme. These inhibitors will act as adjuvants that lack antibacterial activity but can restore
the activity of antibiotics affected by this resistance mechanism, when administered in combination. Cfr
inhibitors will be identified using a target-based approach involving computer-aided screens to be conducted
with virtual libraries of over 120 million commercially available compounds using the Autodock Vina software.
Compounds predicted to bind Cfr will be tested for their ability to bind the purified enzyme in vitro using
Temperature Related Intensity Change measurements and evaluated for their capacity to inhibit Cfr-mediated
methylation of a synthesized rRNA substrate in standard enzyme assays. Promising compounds will then be
tested in an E. coli resistance evolution model to assess the inhibition of Cfr activity in bacterial cells by
monitoring the antibacterial properties of antibiotics whose activity is abolished by the methylation of A2503.
When used in combination, compounds that block the activity of Cfr will result in restored antibiotic activity,
killing the bacterial cells. Inhibitors with this property will be co-crystallized with the enzyme to identify their
binding sites, and potential mechanism of inhibition of Cfr activity. These structural data will also be used in the
design of novel inhibitors with optimized binding affinities and adjuvant properties through fragment-based
drug discovery techniques and structure-activity relationship studies. Optimized adjuvants will be tested against
clinical isolates of bacterial species that employ this form of resistance mechanism.

Grant Number: 5R21AI180902-02
NIH Institute/Center: NIH
Principal Investigator: SQUIRE BOOKER