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Insights into the slow-onset tight-binding inhibition of Escherichia coli dihydrofolate reductase: detailed mechanistic characterization of pyrrolo [3,2-f] quinazoline-1,3-diamine and its derivatives as novel tight-binding inhibitors.

ABSTRACT: 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 Ki value of 7.42 ± 0.92 nm by competitive displacement of the substrate dihydrofolic acid. It shows uncompetitive inhibition vis-à-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 diaminopyrroloquinazoline 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.

SUBMITTER: Srinivasan B

PROVIDER: S-EPMC4445455 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Insights into the slow-onset tight-binding inhibition of Escherichia coli dihydrofolate reductase: detailed mechanistic characterization of pyrrolo [3,2-f] quinazoline-1,3-diamine and its derivatives as novel tight-binding inhibitors.

Srinivasan Bharath B Skolnick Jeffrey J

The FEBS journal 20150306 10

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 inh ...[more]

PMID: 25703118

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Project description:Gram-negative bacteria are implicated in the causation of life-threatening hospital-acquired infections. They acquire rapid resistance to multiple drugs and available antibiotics. Hence, there is the need to discover new antibacterial agents with novel scaffolds. For the first time, this study explores the 1,3,5-triazine-2,4-diamine and 1,2,4-triazine-2,4-diamine group of compounds as potential inhibitors of Escherichia coli DHFR, a pivotal enzyme in the thymidine and purine synthesis pathway. Using differential scanning fluorimetry, DSF, fifteen compounds with various substitutions on either the 3rd or 4th positions on the benzene group of 6,6-dimethyl-1-(benzene)-1,3,5-triazine-2,4-diamine were shown to bind to the enzyme with varying affinities. Then, the dose dependence of inhibition by these compounds was determined. Preliminary quantitative structure-activity relationship analysis and docking studies implicate the alkyl linker group and the sulfonyl fluoride group in increasing the potency of inhibition. 4-[4-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1-yl)phenyl]butyl]benzenesulfonyl fluoride (NSC120927), the best hit from the study and a molecule with no reported inhibition of E. coli DHFR, potently inhibits the enzyme with a Ki value of 42.50 ± 5.34 nM, followed by 4-[6-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1-yl)phenyl]hexyl]benzenesulfonyl fluoride (NSC132279), with a Ki value of 100.9 ± 12.7 nM. Detailed kinetic characterization of the inhibition brought about by five small-molecule hits shows that these inhibitors bind to the dihydrofolate binding site with preferential binding to the NADPH-bound binary form of the enzyme. Furthermore, in search of novel diaminotriazine scaffolds, it is shown that lamotrigine, a 1,2,4-triazine-3,5-diamine and a sodium-ion channel blocker class of antiepileptic drug, also inhibits E. coli DHFR. This is the first comprehensive study on the binding and inhibition brought about by diaminotriazines of a gram-negative prokaryotic enzyme and provides valuable insights into the SAR as an aid to the discovery of novel antibiotics.

Dataset Information

Insights into the slow-onset tight-binding inhibition of Escherichia coli dihydrofolate reductase: detailed mechanistic characterization of pyrrolo [3,2-f] quinazoline-1,3-diamine and its derivatives as novel tight-binding inhibitors.

Publications

Insights into the slow-onset tight-binding inhibition of Escherichia coli dihydrofolate reductase: detailed mechanistic characterization of pyrrolo [3,2-f] quinazoline-1,3-diamine and its derivatives as novel tight-binding inhibitors.

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