Target- and Resistance-Based Mechanistic Studies with TP-434, a Novel Fluorocycline Antibiotic

Abstract

c TP-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. The mechanism of action of TP-434 was assessed using both cell-based and in vitro assays. In Escherichia coli cells expressing recombinant tetracycline resistance genes, the MIC of TP-434 (0.063 g/ml) was unaffected by tet(M), tet(K), and tet(B) and increased to 0.25 and 4 g/ml in the presence of tet(A) and tet(X), respectively. Tetracycline, in contrast, was significantly less potent (MIC > 128 g/ml) against E. coli cells when any of these resistance mechanisms were present. TP-434 showed potent inhibition in E. coli in vitro transcription/translation (50% inhibitory concentration [IC 50 ] ‫؍‬ 0.29 ؎ 0.09 g/ml) and [ 3 H]tetracycline ribosome-binding competition (IC 50 ‫؍‬ 0.22 ؎ 0.07 M) assays. The antibacterial potencies of TP-434 and all other tetracycline class antibiotics tested were reduced by 4-to 16-fold, compared to that of the wild-type control strain, against Propionibacterium acnes strains carrying a 16S rRNA mutation, G1058C, a modification that changes the conformation of the primary binding site of tetracycline in the ribosome. Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis and that this activity is largely unaffected by the common tetracycline resistance mechanisms.

Structural basis for TetM-mediated tetracycline resistance

“…In contrast, the attached C9-glycyl side chain of Tgc would prevent access of the residues of loop III of TetM (Fig. 3 E and F), leading us to suggest that this steric hindrance contributes, together with the increased affinity of Tgc (7,8,27), to explaining how Tgc overcomes TetM-mediated resistance whereas tetracycline cannot (8). Curiously, loop III of EF-G contains a highly conserved histidine residue (H583; SI Appendix, Fig.…”

“…The TetM homology model and ribosome crystal structures (18,19,35,36) were fitted as rigid bodies to the cryo-EM density by using Coot (37) and Chimera (38). The QuikChange mutagenesis kit (Qiagen) was used to introduce sitespecific mutations into the tetM gene according to the manufacturer's instructions, and minimal inhibitory concentrations were determined as described previously (8). Detailed materials and methods can be found in the SI Appendix, Materials and Methods.…”

“…S10 I-M). Given that the TetM•70S complex was formed with 10 μM Tgc, which is ∼100 times its IC 50 for in vitro translation (8), and that Tgc has a ∼10-fold higher affinity for the ribosome than tetracycline (7,8,27), the absence of secondary binding sites illustrates the increased specificity of Tgc compared with tetracycline.…”

“…Of the variety of tetracycline-specific resistance mechanisms, efflux and ribosome-protection are the most common (5). The third generation of tetracycline derivatives, such as tigecycline (Tgc), display enhanced antimicrobial activity, overcoming efflux and ribosome protection mechanisms (6)(7)(8).…”

Antibacterial Activity of Eravacycline (TP-434), a Novel Fluorocycline, against Hospital and Community Pathogens

“…Eravacycline is a novel fluorocycline antibiotic designed to overcome resistance to common tetracycline-specific efflux and ribosomal protection mechanisms and is impervious to other antibiotic-specific resistance mechanisms (14)(15)(16)(17). Similar to other members of the tetracycline antibiotic class, eravacycline has been shown to be a potent, mechanism-based inhibitor of the bacterial ribosome (16).…”