Journal
FEBS JOURNAL
Volume 282, Issue 10, Pages 1922-1938Publisher
WILEY-BLACKWELL
DOI: 10.1111/febs.13244
Keywords
dihydrofolate reductase; drug discovery; mechanistic characterization; pyrrolo [3,2-f] quinazoline-1,3-diamine; slow-tight-binding inhibition
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Funding
- Division of General Medical Sciences of the NIH [GM-37408, GM-48835]
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Dihydrofolate reductase (DHFR) is a pivotal enzyme involved in the de novo pathway of purine synthesis, and hence, represents an attractive target to disrupt systems that require rapid DNA turnover. The enzyme acquires resistance to available drugs by various molecular mechanisms, which necessitates the continuous discovery of novel antifolates. Previously, we identified a set of novel molecules that showed binding to E. coli DHFR by means of a thermal shift without establishing whether they inhibited the enzyme. Here, we show that a fraction of those molecules represent potent and novel inhibitors of DHFR activity. 7-[(4-aminophenyl) methyl]-7H-pyrrolo [3,2-f] quinazoline-1,3-diamine, a molecule with no reported inhibition of DHFR, potently inhibits the enzyme with a K-i value of 7.42 +/- 0.92 nM by competitive displacement of the substrate dihydrofolic acid. It shows uncompetitive inhibition vis-a-vis NADPH, indicating that the inhibitor has markedly increased affinity for the NADPH-bound form of the enzyme. Further, we demonstrate that the mode of binding of the inhibitor to the enzyme-NADPH binary complex conforms to the slow-onset, tight-binding model. By contrast, mechanistic characterization of the parent molecule 7H-pyrrolo [3,2-f] quinazoline-1,3-diamine shows that lack of (4-aminophenyl)-methyl group at the seventh position abolishes the slow onset of inhibition. This finding provides novel insights into the role of substitutions on inhibitors of E. coli DHFR and represents the first detailed kinetic investigation of a novel diaminopyrrolo-quinazoline derivative on a prokaryotic DHFR. Furthermore, marked differences in the potency of inhibition for E. coli and human DHFR makes this molecule a promising candidate for development as an antibiotic.
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