Identification of an antibiotic from an HTS targeting EF-Tu:tRNA interaction: a prospective topical treatment for MRSA skin infections

Abstract

Because of the urgent need for new antibiotics to treat drug-resistant bacterial pathogens, we employed an assay that rapidly screens large quantities of compounds for their ability to interfere with bacterial protein synthesis, in particular, the delivery of amino acids to the ribosome via tRNA and elongation factor Tu (EF-Tu). We have identified a drug lead, named MGC-10, which kills Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), with a MIC of 6 µM, while being harmless to mammalian cells in vitro in that concentration range. The antibacterial activity of MGC-10 was broad against over 50 strains of antibiotic-resistant samples obtained from hospital infections, where MGC-10 inhibited all tested strains of MRSA. Extensive selection and screening with MGC-10 did not yield any resistant strains, indicating it may have universal antibacterial activity against S. aureus. Pharmacokinetics performed in mice suggested that MGC-10 was too toxic for systemic use; however, it appears to have potential as a topical treatment for difficult-to-treat wounds or skin infections by Gram-positive pathogens such as MRSA. In a mouse skin-infection model with MRSA, MGC-10 performed as well or better than the present topical drug of choice, mupirocin. MGC-10 showed little, if any, accumulation in the livers of topically treated mice. These results bode well for the future use of MGC-10 in clinical application as it could be used to treat a broad range of S. aureus skin infections that are resistant to known antibiotics.IMPORTANCEThere is a critical need for new antibiotics to treat bacterial infections caused by pathogens resistant to many if not all currently available antibiotics. We describe here the identification of a prospective new antibiotic from high-throughput screening of a chemical library. The screening was designed to detect the inhibition of formation of a complex required for bacterial protein synthesis in all bacteria, the "ternary complex," comprised of elongation factor Tu (EF-Tu), aminoacyl-tRNA, and GTP. The inhibitory compound, renamed MGC-10, was effective against all Gram-positive bacteria, including a wide variety of methicillin-resistant Staphylococcus aureus (MRSA) strains. Although apparently too toxic for systemic use, the compound was safe and effective for topical use for treating skin infections in a mouse model. No resistance to the compound has been detected thus far, suggesting the potential to develop this compound for topical use to treat infections, especially those caused by pathogens resistant to existing antibiotics.

Keywords: (R,R)-tetrahydrochrysene; FRET; Staphylococcus aureus; ternary complex in protein synthesis; topical antibiotic for Gram-positive pathogens.

Fig 1

Ternary complex involving EF-Tu and tRNA with two engineered fluorescent labels, Cy3 and Cy5 for the FRET assay (3).

Fig 2

Inhibition of FRET by known inhibitors of EF-Tu; M, molarity. Shown is a representative experiment, which was repeated.

Fig 3

LOPAC1280 results for the offline and online assays. FRET values were normalized to DMSO controls (0%) and 40 µM kirromycin (−100%). Red dots represent compounds that showed large discrepancies between the two methods. The green dot represents MGC-10.

Fig 4

Secondary FRET assay. Controls are represented by two bars on the left side. Cy5 acceptor fluorescence intensity was significantly diminished in the presence of the inhibitor of the reaction, MGC-10. The gray and green bars represent controls of each dye alone attached to EF-Tu or tRNA, respectively (background fluorescence), and the two dyes together without MGC-10 added (FRET). The red bars show the extent of fluorescence in the presence of increasing amounts of MGC-10.

Fig 5

(Left) (R,R)-tetrahydrochrysene chemical structure. (Right) Numbering of carbon atoms in chrysene.

Fig 6

Inhibition of B. subtilis growth by MGC-10. The green bar represents growth in the absence of an inhibitor. The red bars represent significant growth inhibition by MGC-10.

Fig 7

Testing of antibacterial properties of nine derivatives of (R,R)-tetrahydrochrysene (MGC-10). The boxed numbers represent the thickness in mm of zones of inhibition around filter disks containing the respective compounds (see Materials and Methods).

Fig 8

Effects of varying concentrations of MGC-10 on the efficiency of ribosomal translation in vitro. (A) Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) following in vitro translation with the PURExpress translation kit (New England Biolabs). The protein synthesized (Dihydrofolate reductase) in the reaction is shown in the small rectangles superimposed on the gel. (B) Quantification using a BioRad gel scanner and software provided with this scanner. The density of the image in areas highlighted by the small rectangles on the gel was measured and shown as a function of added MGC-10 concentration.

Fig 9

Bactericidal test of MGC-10 on S. aureus. Cell survival is shown as a function of time following exposure to MGC-10 or to DMSO (control).

Fig 10

MGC-10 reduces S. aureus-induced dermonecrosis. Mice were infected subcutaneously with S. aureus USA300 for 5 days. (A) Area of dermonecrosis measured over time. Lines display mean and error. Statistical significance between the untreated control (ointment without MGC-10) and MGC-10 treated mice. (B) Bacterial concentration in punch biopsies at the conclusion of the experiment. n = 18 untreated and n = 17 mupirocin and MGC-10. Each point represents a mouse. Lines display median. **P < 0.01 and *P < 0.05.