title: Design, synthesis, and evaluation of 9-D-ribitylamino-1,3,7,9-tetrahydro-2,6,8-purinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents as inhibitors of tiboflavin synthase and lumazine synthase.
abstract: Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport processes. To obtain structural probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a series of ribitylpurinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents were synthesized. Since the purinetrione ring system and the ribityl hydroxyl groups can be alkylated, the synthesis required the generation of these two moieties in protected form before the desired alkylation reaction could be carried out. These substances were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site. All of the compounds were found to be effective inhibitors of both Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of 3-(1,3,7,9-tetrahydro-9-D-ribityl-2,6,8-trioxopurin-7-yl)propane 1-phosphate provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. Interestingly, the enzyme kinetics of these new compounds in comparison with the parent purinetrione demonstrated unexpectedly that the phosphate and phosphonate substituents contributed negatively to the binding. A possible explanation for these effects on lumazine synthase would be that the inorganic phosphate in the assay buffer competes with the substituted purinetriones for binding to the enzyme. This would be consistent with the observed increase in K(m) of the 3,4-dihydroxybutanone-4-phosphate substrate from 5.2 microM in Tris buffer or from 6.7 microM in MOPS buffer to 50 microM in phosphate buffer when tested on Bacillus subtilis lumazine synthase. However, when tested in Tris buffer vs Mycobacterium tuberculosis lumazine synthase, three of the phosphate inhibitors displayed inhibition constants in the 4-5 nM range, indicating that they are much more potent than the parent purinetrione. Under these conditions, the phosphate moieties of the inhibitors do contribute positively to their binding. The alpha,alpha-difluorophosphonate analogue, which is expected to have enhanced metabolic stability relative to the phosphates, was also found to be an inhibitor of Mycobacterium tuberculosis lumazine synthase with a K(i) of 60 nM.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Cushman
authors.forename: Thota
authors.initials: T
authors.lastname: Sambaiah
authors.forename: Guangyi
authors.initials: G
authors.lastname: Jin
authors.forename: Boris
authors.initials: B
authors.lastname: Illarionov
authors.forename: Markus
authors.initials: M
authors.lastname: Fischer
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D001667:Binding, Competitive; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000235:genetics; D010755:Organophosphates / Q000737:chemistry / Q000494:pharmacology; D063065:Organophosphonates / Q000737:chemistry / Q000494:pharmacology; D011687:Purines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D012255:Ribitol / Q000031:analogs & derivatives / Q000494:pharmacology; D012258:Riboflavin Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D010755:Organophosphates; D063065:Organophosphonates; D011687:Purines; D011994:Recombinant Proteins; D012255:Ribitol; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012258:Riboflavin Synthase