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

Antibiotics that operate 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 , , and , among other organisms. Through X-ray crystallography, we confirmed engagement of the DHFR target and found that some DADHTs stabilize a previously unobserved conformation of the enzyme but, broadly, bind in the occluded conformation. Using inhibition of purified and DHFR and disruption of 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.