# Structural basis of Cfr-mediated resistance to antibiotics targeting the bacterial ribosome

> **NIH NIH R21** · UNIVERSITY OF ILLINOIS AT CHICAGO · 2021 · $232,788

## Abstract

SUMMARY
The ribosome is an essential drug target as many classes of clinically important antibiotics bind to its functional
centers and interfere with different aspects of protein synthesis. Out of several functional centers, the catalytic
peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical
classes. One of the most abundant and clinically relevant mechanisms of resistance to such PTC-acting drugs
is based on the methylation of the C8-position of an adenine residue 2503 (A2503) of the 23S rRNA by Cfr
methyltransferase. This modification confers resistance to a wide range of PTC-targeting antibiotics, including
phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramins A (PhLOPSa phenotype), as well
as 16-membered macrolides and hygromycin A. The Cfr-mediated drug resistance is an alarming healthcare
threat because of the rapid spread of the cfr gene among pathogenic bacteria renders many clinically important
antibiotics ineffective for anti-infection therapies. Thus, developing next-generation PTC-targeting inhibitors
acting against the Cfr-positive pathogens is of utmost medical importance. While this problem could be tackled
by structure-driven rational drug design, a structure of the Cfr-modified ribosome is currently unknown.
In the proposed project, we will solve this problem by determining the first high-resolution X-ray crystal structure
of the Cfr-modified bacterial ribosome in isolation or in complex with antibiotics exhibiting residual activity against
the A2503-methylated ribosome. To achieve this goal, we will optimize the expression of functionally-active Cfr
methyltransferases in the cells of thermophilic bacterium Thermus thermophilus, which has been widely used as
the source of ribosomes suitable for crystallographic studies. We have found that in spite of growing optimally at
65-72°C, T. thermophilus can also grow at notably lower temperatures (48°C). By cloning and expressing Cfr-
methyltransferase genes from moderately thermophilic bacteria in T. thermophilus at high temperatures (65-
72°C) or by expressing the erm genes from the mesophilic bacteria in the T. thermophilus host grown at reduced
temperatures (48-65°C), we will obtain crystallizable ribosomes with methylated A2503. Once the structure of
the A2503-methylated ribosome is determined, we will solve the structures of this Cfr-modified ribosome in
complex with several PTC-acting drugs exhibiting residual binding affinity for the modified ribosome. The
resulting information will be instrumental for understanding the molecular principles of Cfr-mediated resistance
and will instruct the subsequent rational design of new antibacterials active against Cfr-positive pathogens.
Moreover, upon successful completion of the proposed project, we shall answer a long-standing fundamental
and medically relevant question: How the addition of a methyl group to a single nucleotide in the 2.5-MDa
ribosome results ...

## Key facts

- **NIH application ID:** 10282911
- **Project number:** 1R21AI163466-01
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT CHICAGO
- **Principal Investigator:** YURY POLIKANOV
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $232,788
- **Award type:** 1
- **Project period:** 2021-06-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10282911

## Citation

> US National Institutes of Health, RePORTER application 10282911, Structural basis of Cfr-mediated resistance to antibiotics targeting the bacterial ribosome (1R21AI163466-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10282911. Licensed CC0.

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