Crystal Structure of Lumazine Synthase from Mycobacterium tuberculosis as a Target for Rational Drug Design:  Binding Mode of a New Class of Purinetrione Inhibitors^,

Abstract

The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range.

Lumazine Synthase from Candida albicans as an Anti-fungal Target Enzyme

“…The energy minimization procedure turned the indole ring of Trp-27 to a parallel conformation with respect to the plane of the aromatic groups of all inhibitors, whereas the indole group in the empty structure assumed different conformations in different subunits. This conformational change of the side chain of Trp-27 is in good agreement with the conformations of the aromatic groups of tryptophan or phenylalanine found in the structures of different LSs inhibitor complexes (9,10,12,18,49,52,53). The main-chain contacts, found conserved in all orthologous complexes, remained unchanged in the binding models of all inhibitors.…”

“…(20,21,48). It has been shown that the purinetrione ring system with an attached C5-phosphate side chain resulted in a less potent inhibitor of B. subtilis LS (20); however, it revealed a high inhibition potential for the pentameric enzyme from M. tuberculosis (12,20,49). The present investigation of binding processes has shown that the values of the association constants of those inhibitors to CALS are in a similar range compared with those for M. tuberculosis LS.…”

“…According to these facts we have undertaken docking studies of inhibitors to the active site and we present in this report the hypothetical binding modes for complexes of CALS with all four inhibitors described above. Calculations carried out with Autodock for compound TS44 revealed all obtained 50 models in the same position inside the active site corresponding to the binding mode of this compound in the MbtLS/TS44 structure (12) (Fig. 7a).…”

Antitubercular Drugs Development: Recent Advances in Selected Therapeutic Targets and Rational Drug Design

“…Two enzymes involved in the de novo biosynthesis of NAD that affects the NADH/NAD + ratio upon which M. tuberculosis is dependent, have been studied as possible drug targets (Bellinzoni et al, 2002). Genomic analysis studies have suggested that the riboflavin biosynthesis pathway is essential in M. tuberculosis (Morgunova et al, 2005) and the lumazine synthase pathway has been validated as a target for anti-TB drug discovery.…”

In-Silico Structure Based Drug Design of a Potent Inhibitor of Enzyme Lumazine Synthase- A Novel Therapeutic Target for Tuberculosis

“…Figure.1 A schematic diagram of terminal reactions that are catalyzed by lumazine synthase and riboflavin synthase in the pathway of riboflavin biosynthesis. (A) 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; (B) 3,4-dihydroxy-2-butanone 4-phosphate; (C) 6,7-dimethyl-8-ribityllumazine; and (D) riboflavin So far various pyrimidine or purine ring containing compounds were reported as potent inhibitors of LS (Persson et al, 1999;Gerhardt et al, 2002;Morgunova et al, 2005;Morgunova et al, 2006;Zhang, Y. et al, 2008;Zhang, X. et al, 2008). These compounds were almost identical to substrate of LS (designated as A in figure1) and inhibit the activity of the enzyme in competitive mechanism.…”