Gram-Negative Antibiotic Active Through Inhibition of an Essential Riboswitch

Abstract

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

Figure 1.

Ribocil C as a starting point to access a GN-active antimicrobial that engages the essential FMN riboswitch. A) Accumulation in E. coli MG1655 and Minimum Inhibitory Concentration (MIC) values against strains. MICs were performed in M9-MOPS media per CLSI guidelines. B) Location prioritized for amine installation, based on the co-crystal of Ribocil B bound to the FMN riboswitch aptamer of F. nucleatum (PDB 5C45). C) Target compounds designed to have enhanced accumulation in GN bacteria.

Figure 2.

eNTRy rule guided design of amine analogues of Ribocil C that accumulate within and kill GN pathogens. A) Workflow utilized to develop new Ribocil C variants that possess activity against wild-type GN pathogens. B) Lead compounds identified with the ability to accumulate in wild-type E. coli and inhibit growth. Antimicrobial assessment of lead derivatives against various GN pathogens. MC E. coli: Membrane Compromised E. coli. Accumulation in E. coli MG1655, units are nmol/10¹² CFUs. MICs were performed in M9-MOPS media per CLSI guidelines.

Figure 3.

Synthetic modifications of compound 3 to probe the effect of functional groups on antimicrobial activity. Accumulation was performed in E. coli MG1655 and is represented in nmol/10¹² CFUs. The accumulation of compound 6 could not be determined due to limitations of aqueous solubility. The MIC values for all compounds are reported for the racemates. The accumulation values for all compounds are reported for the racemates, with the exception of compound 3, which is reported for (-)3. MIC values were determined for E. coli BW25113 (WT E. coli), E. coli JW3596 (E. coli ΔrfaC), and E. coli JW5503 (E. coli ΔtolC). MICs were performed in M9-MOPS media per CLSI guidelines.

Figure 4.

Assessment of Ribocil C-PA and Ribocil C against MDR clinical isolates of A) E. coli (n=42) and B) K. pneumoniae (n=54). MICs were performed in M9-MOPS media per CLSI guidelines. See Supporting Table 3 for list of resistance genes in clinical isolate panels. A full listing of this MIC data is in Supporting Tables 4–5.

Figure 5.

Flavin reduction in E. coli BW25113 at sub-inhibitory concentration (2 μg/mL) of Ribocil C-PA. The inactive enantiomer, (+)3, was also assessed at 2 μg/mL and demonstrates no inhibition of flavin production. Flavin monitoring was performed after cultures were treated with either vehicle or compound for 20 hours. Data are shown as means ± s.d. and statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons. NS, not significant (P> 0.05). *P< 0.015).

Figure 6:

Mutations observed after sequencing the FMN riboswitch in Ribocil C-PA resistant clones of E. coli BW25113 generated at 8X the MIC (32 μg/mL) of Ribocil C-PA.

Figure 7.

A) Bacterial burden model of acute pneumonia in E. coli. Acute pneumonia infections initiated in seven-week-old male CD-1 mice with E. coli AR0493 (1.7×10⁹ CFU mouse⁻¹ intranasally) or E. coli ELZ4081 (3.1×10⁹ CFU mouse⁻¹ intranasally). Mice were treated with vehicle (n=8) or compound (Ribocil C or Ribocil C-PA, 100 mg/kg IP, n=8 for each group) 8, 16, 30 and 48h post-infection, and the bacterial burden was evaluated 48h post-infection. MIC=8 μg/mL and 4 μg/mL for Ribocil C-PA against E. coli AR0493 and E. coli ELZ4081 respectively. B) Kaplan-Meier survival curve for the mouse efficacy model of E. coli sepsis. Seven-week-old male CD-1 mice were infected with E. coli AR0493 (7.91×10⁷ CFU mouse⁻¹; 15 mice per group) via intravenous injection. Mice were treated with compound (Ribocil C or Ribocil C-PA, 100 mg/kg IP, n=15 for each group) twice daily post-infection for 5 days. In A, data are shown as means ± s.d. and statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons. NS, not significant (P> 0.05), ****P< 0.0001. In B, statistical significance was determined by two-tailed log-rank (Mantel–Cox) test, ***P<0.001.