A New Series of N-[2,4-Dioxo-6-d-ribitylamino-1,2,3,4-tetrahydropyrimidin-5-yl]oxalamic Acid Derivatives as Inhibitors of Lumazine Synthase and Riboflavin Synthase:  Design, Synthesis, Biochemical Evaluation, Crystallography, and Mechanistic Implications

Abstract

The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.

Virtual screening, selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase

“…Comparison of these models with the known structures of M. tuberculosis lumazine synthase in complex with different substrate 1 analogues showed the lack of five hydrogen bonds involving the ribityl chains of the substrate 1 analogues,43,54 and at least three hydrogen bonds formed between oxygen/nitrogen atoms of heterocyclic rings of the substrate 1 analogues and main/side chain atoms of Val81, Ile83 and Trp27 (Figure 3). Trp27 is involved in binding through π-π stacking interactions.…”

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

“…All rotatable bonds of ligands were set to be rotatable. The 40 x 40 x 40 dimension of grid box size and 0.375 Å grid spacing was placed around the catalytic triad to cover the entire enzyme active site mentioned by Zhang, Y. et al, (2008) and accommodate ligands to move freely. Clustering histogram analyses were performed after the docking searches.…”

“…The result of in-silico ADMET study of ligand Ex1 Zhang, Y. et al, (2008) mentioned that the active site of Mycobacterium tuberculosis Lumazine Synthase (LS) is located at the interface between two neighboring subunits in the pentameric assembly. The active site is consists of the residues from three β-loops (residues 26-28, 58-61, 81-87) of one subunit as well as residues 128-141 and residue Asn114 of the neighboring subunit.…”

“…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.…”

15N{31P} REDOR NMR Studies of the Binding of Phosphonate Reaction Intermediate Analogues to Saccharomyces cerevisiae Lumazine Synthase

“…That effort eventually resulted in the preparation of the intermediate analogue 13 and the crystal structure displayed in Figure 1, in which the ligand 13 is bound to Mycobacterium tuberculosis lumazine synthase in an extended conformation resembling that proposed for intermediate 6 in Scheme 2. The amide carbonyl of 13 was found to be hydrogen bonded to water, which in turn was hydrogen bonded to the enzyme-bound inorganic phosphate (25). …”