Expanding the Landscape of Dual Action Antifolate Antibacterials through 2,4-Diamino-1,6-dihydro-1,3,5-triazines

Abstract

Antibiotics that operate via multiple mechanisms of action are a promising strategy to combat growing resistance. Previous studies have shown that dual action antifolates formed from a pyrroloquinazolinediamine core can inhibit the growth of bacterial pathogens without developing resistance. In this work, we expand the scope of dual action antifolates by repurposing the 2,4-diamino-1,6-dihydro-1,3,5-triazine (DADHT) cycloguanil scaffold to a variety of derivatives designed to inhibit dihydrofolate reductase (DHFR) and disrupt bacterial membranes. Dual mechanism DADHTs have activity against a variety of target pathogens, including Mycobacterium tuberculosis, Mycobacterium abscessus, and Pseudomonas aeruginosa, among other ESKAPEE organisms. Through X-ray crystallography, we confirmed engagement of the Escherichia coli DHFR target and found that some DADHTs stabilize a previously unobserved conformation of the enzyme but, broadly, bind in the occluded conformation. Using in vitro inhibition of purified E. coli and Staphylococcus aureus DHFR and disruption of E. coli membranes, we determined that alkyl substitution of dihydrotriazine at the 6-position best optimizes the DADHT's two mechanisms of action. By employing both mechanisms, the DADHT spectrum of activity was extended beyond the scope of traditional antifolates. We are optimistic that the dual mechanism approach, particularly through the action of antifolates, offers a unique means of combating hard-to-treat bacterial infections.

Keywords: antibiotic; antifolate; dihydrofolate reductase; dual mechanism; membrane disruption; triazine.

Figure 1.

Chemical structures of current antimicrobial antifolates and general structure and mechanism for dual action antifolates.

Figure 2.. X-ray crystal structures of EcDHFR bound to cycloguanil (CYC) and compound 3d.

a and d show the cartoon depiction of EcDHFR with the respective ligands (CYC and 3d) bound. b and e show the surface depiction of EcDHFR with the respective ligands (CYC and 3d) bound. c shows the polar contacts between compound CYC and the active site of EcDHFR. f shows the polar contacts between 3d and the active site of EcDHFR.

Figure 3.. 6-(nonyl) and 6-(undecyl) substitutions show significant membrane disruption in E. coli.

a and b show the mean of three independent trials measuring fluorescence intensity at 520 nm upon treatment of E. coli ATCC 25922 cells with compounds 3d and 3e, respectively, at 0 min. Data points above the solubility limits of each compound in PBS are not shown. Full datasets can be found in Figure S39. c shows a plot of 1/(MIC₉₀) vs clogP calculated using ChemDraw Professional v21. MIC₉₀ data for IRS-16 was sourced from the literature. MIC₉₀ data for all other compounds was sourced from Table 1. Compounds with MIC₉₀ > 128 μM were excluded.

Figure 4.

Summary structure-activity relationships of hydrophobic cycloguanil derivatives.

Scheme 1.

Synthesis of N1-aryl-substituted DADHTs.

Scheme 2.

Synthesis of C6-substituted DADHTs.

Scheme 3.

Structures of additional compounds synthesized.