Compound Uptake into E. coli Can Be Facilitated by N-Alkyl Guanidiniums and Pyridiniums

Abstract

Multidrug-resistant Gram-negative bacterial infections are on the rise, and with no FDA approvals for new classes of broad-spectrum antibiotics in over 50 years, these infections constitute a major threat to human health. A significant challenge is the inability of most compounds to accumulate in Gram-negative bacteria. Recently developed predictive guidelines show that appending a primary amine to an appropriately shaped compound can enhance Gram-negative accumulation. Here, we report that other positively charged nitrogen functional groups, namely, N-alkyl guanidiniums and pyridiniums, can also facilitate compound uptake into Gram-negative bacteria. The accumulation of a set of 60 nonantibiotic compounds, consisting of 20 primary amines and their corresponding guanidiniums and pyridiniums, was assessed in Escherichia coli. We also installed these alternate functional groups onto antibiotic scaffolds and assessed their accumulation and antibacterial activity in Gram-negative bacteria. The results suggest that other positively-charged, nitrogen-containing functional groups should be considered when designing antibiotics with Gram-negative activity.

Keywords: Gram-negative accumulation; amines; antibiotic drug design; guanidiniums; pyridiniums.

Figure 1.

eNTRy rules have been used to enhance the Gram-negative accumulation and antibacterial activity of Gram-positive only antibiotics in multiple contexts. 6DNM, Debio-1452, Ribocil C, AZ17, Pyrrole 8v, and CDCHD-FuA. Accumulation measured in nmol/10¹² CFUs in E. coli MG1655.

Figure 2.

(A) Functional groups with ionizable nitrogens and their electrostatic representations. (B) Some FDA approved drugs containing primary alkyl guanidiniums and alkylpyridiniums.

Figure 3.

(A) 20 primary amines evaluated for accumulation in E. coli, along with their guanidinium and pyridinium derivatives. (B) A two-step guanidinylation or Zincke reaction was used in most cases to construct the corresponding guanidinium and pyridinium, respectively. (C) Summary of accumulation of these 60 compounds in E. coli MG1655. Compounds are categorized as accumulators or non-accumulators on the basis of statistical significance relative to the negative controls; see the Methods and Supporting Information for full details.

Figure 4.

Properties affecting accumulation of alkyl guanidiniums and pyridiniums in E. coli. (A) Amphiphilic moment (vsurf_A) is correlated with guanidinium vs primary amine accumulation within structural classes. (B) A higher number of rotatable bonds does not prevent the accumulation of certain guanidiniums. (C) Structure and properties of accumulating pyridiniums. All experiments were performed in biological triplicate. Accumulation measured in nmol/10¹² CFUs in E. coli MG1655.

Figure 5.

Charge delocalization and sum of HBD strengths are important for the accumulation of amines vs guanidiniums vs pyridiniums for the 60 compounds described in Figure 3. (A) ESPMAX and (B) h_emd correlate with the overall accumulation trends. (C) Accumulation across a structural series decreases as ESPMAX and h_emd decrease.

Figure 6.

Accumulation and activity of antibiotics with appended amines, guanidiniums, and pyridiniums. All experiments were performed in biological triplicate. Accumulation measured in nmol/10¹² CFUs in E. coli MG1655 after a 10 min incubation with the exception of the Debio derivatives (21–24, in which accumulation was assessed after a 2 h incubation). MICs were performed in MH broth or M9-MOPS media per Clinical and Laboratory Standards Institute (CLSI) guidelines in S. aureus 29213, E. coli MG1655, and E. coli JW5503 ΔtolC and reported in μg/mL. n/a = not applicable. n.d. = not determined due to the lack of solubility at the assay concentrations. ^{a, b, c}Data previously reported.^,,

Figure 7.

(A) Structures of preclinical antibiotics and adjuvants bearing guanidinium motifs.^,, (B) Calculated ESPMAX ranges for different positively charged functional groups on nonantibiotic scaffolds.