Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I

Abstract

DNA ligases are important enzymes which catalyze the joining of nicks between adjacent bases of double-stranded DNA. NAD+-dependent DNA ligases (LigA) are essential in bacteria and are absent in humans. They have therefore been identified as novel, validated and attractive drug targets. Using virtual screening against an in-house database of compounds and our recently determined crystal structure of the NAD+ binding domain of the Mycobacterium tuberculosis LigA, we have identified N1, Nn-bis-(5-deoxy-α-d-xylofuranosylated) diamines as a novel class of inhibitors for this enzyme. Assays involving M.tuberculosis LigA, T4 ligase and human DNA ligase I show that these compounds specifically inhibit LigA from M.tuberculosis. In vitro kinetic and inhibition assays demonstrate that the compounds compete with NAD+ for binding and inhibit enzyme activity with IC50 values in the µM range. Docking studies rationalize the observed specificities and show that among several glycofuranosylated diamines, bis xylofuranosylated diamines with aminoalkyl and 1, 3-phenylene carbamoyl spacers mimic the binding modes of NAD+ with the enzyme. Assays involving LigA-deficient bacterial strains show that in vivo inhibition of ligase by the compounds causes the observed antibacterial activities. They also demonstrate that the compounds exhibit in vivo specificity for LigA over ATP-dependent ligase. This class of inhibitors holds out the promise of rational development of new anti-tubercular agents.

Evaluation of NAD + -Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

“…It is this distribution of cofactor specificity that has led to the suggestion that NAD ϩ -dependent DNA ligases may be exploited as useful new targets for broad-spectrum antibacterial compounds (5,24,29,32). Indeed, recent studies have begun to make important progress in identifying small molecules that have some specificity towards the inhibition of NAD ϩ -dependent DNA ligases (2,(26)(27)(28).Although NAD ϩ -dependent DNA ligases appear to be produced in all bacteria, some bacteria encode additional ATPdependent versions of the proteins (5,24,29,32). This complicates potential strategies to target NAD ϩ -dependent DNA ligases with antibiotics, as it is not clear whether the ATPdependent enzymes would influence the efficacy of any compound.…”

“…It is this distribution of cofactor specificity that has led to the suggestion that NAD ϩ -dependent DNA ligases may be exploited as useful new targets for broad-spectrum antibacterial compounds (5,24,29,32). Indeed, recent studies have begun to make important progress in identifying small molecules that have some specificity towards the inhibition of NAD ϩ -dependent DNA ligases (2,(26)(27)(28).…”

Mechanistic Assessment of DNA Ligase as an Antibacterial Target in Staphylococcus aureus

“…These effects can be illuminated by examination of the available crystal structures of LigA adenylation domains from several pathogenic bacteria, some featuring bound ligands, including substrate, product, and/or various inhibitors (Fig. 6A) (8,12,19,27; also unpublished data of C. Pinko, A. Borchardt, V. Nikulin, and Y. Su). Most structures show an "open" orientation of domain Ia relative to the NTase domain, with two well-separated (Ͼ20 Å) ligand-binding sites at the domain Ia surface and the NTase active site.…”

“…Numerous LigA inhibitors have been reported to date, including arylamino acids, such as chloroquine (4), glycosyl ureides and glycosylamines (27,28), tetracyclic indoles (29), a pyrimidopyrimidine inhibitor (17), substituted adenosine analogs (19,30), and the pyridochromanones (1). Pyridochromanones were identified by high-throughput screening as potent competitive inhibitors of DNA ligation by LigA from Staphylococcus aureus (50% inhibitory concentration [IC 50 ] Յ 0.9 M) (1).…”

“…The subsequent coupling of adenylation to DNA ligation depends upon downstream DNA-binding domains, which include an oligonucleotide-binding fold (OB fold) and a helix-hairpin-helix (HhH) domain. Structural studies of the adenylation domain have revealed conformational transitions that accompany the adenylation cycle (8), and structural study of the full-length enzyme bound to DNA-adenylate has identified specific contacts between the DNA-binding domains and the DNA duplex substrate near the nicked ligation site (20).Numerous LigA inhibitors have been reported to date, including arylamino acids, such as chloroquine (4), glycosyl ureides and glycosylamines (27,28), tetracyclic indoles (29), a pyrimidopyrimidine inhibitor (17), substituted adenosine analogs (19,30), and the pyridochromanones (1). Pyridochromanones were identified by high-throughput screening as potent competitive inhibitors of DNA ligation by LigA from Staphylococcus aureus (50% inhibitory concentration [IC 50 ] Յ 0.9 M) (1).…”