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

DNA ligases are important enzymes which catalyze the joining of nicks between adjacent bases of double-stranded DNA. NAD1-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 NAD1 binding domain of the Mycobacterium tuberculosis LigA, we have identified N1, Nn-bis-(5-deoxy-a-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 NAD1 for binding and inhibit enzyme activity with IC50 values in the mM 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 NAD1 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 ATPdependent ligase. This class of inhibitors holds out the promise of rational development of new anti-tubercular agents.

Mechanistic insights from the crystal structures of a feast/famine regulatory protein from Mycobacterium tuberculosis H37Rv

Rv3291c gene from Mycobacterium tuberculosis codes for a transcriptional regulator belonging to the (leucine responsive regulatory protein/regulator of asparigine synthase C gene product) Lrp/AsnC-family. We have identified a novel effectorbinding site from crystal structures of the apo protein, complexes with a variety of amino acid effectors, X-ray based ligand screening and qualitative fluorescence spectroscopy experiments. The new effector site is in addition to the structural characterization of another distinct site in the protein conserved in the related AsnC-family of regulators. The structures reveal that the ligandbinding loops of two crystallographically ndependent subunits adopt different conformations to generate two distinct effector-binding sites. A change in the conformation of the binding site loop 100–106 in the B subunit is apparently necessary for octameric association and also allows the loop to interact with a bound ligand in the newly identified effector-binding site. There are four sites of each kind in the octamer and the protein preferentially binds to aromatic amino acids. While amino acids like Phe, Tyr and Trp exhibit binding to only one site, His exhibits binding to both sites. Binding of Phe is accompanied by a conformational change of 3.7A ° in the 75–83 loop, which is advantageously positioned to control formation of higher oligomers. Taken together, the present studies suggest an elegant control mechanism for global transcription regulation involving binding of ligands to the two sites, individually or collectively.

C-3 Alkyl /Arylalkyl-2,3-dideoxy hex-2-enopyranosides as Antitubercular Agents: Synthesis, Biological Evaluation and QSAR Study

A series of C-3 alkyl and arylalky 2,3-dideoxy hex-2-enopyranoside derivatives were synthesized by Morita-Baylis-Hillman reaction using enulosides 4, 5 and 6 and various aliphatic and aromatic aldehydes. The compounds were evaluated in vitro for the complete inhibition of growth of Mycobacterium tuberculosis H37Rv. They exhibited moderate to good activity in the range of 25-1.56 µg/mL. Among these, 4d, 4h, 5c and 4hr showed activity at minimum inhibitory concentrations, 3.12, 6.25, 1.56 and 1.56µg/mL, respectively. These compounds were safe against cytotoxicity in VERO cell line and mouse macrophage cell line J 744A.1. A QSAR analysis by CP-MLR with alignment-free 3D-descriptors indicated the relevance of structure space comparable to the minimum energy conformation (from conformational analysis) of 5c to the activity. The study indicates that the compounds attaining conformational space 5c and reflecting some symmetry, minimum eccentricity and closely placed geometric and electronegativity centers therein are favorable for activity.