Identification of novel isocytosine derivatives as xanthine oxidase inhibitors from a set of virtual screening hits
Chandrika B-Rao, Asha Kulkarni-Almeida, Kamlesh V. Katkar, Smriti Khanna, Usha Ghosh, Ashish Keche, Pranay Shah, Ankita Srivastava, Vaidehi Korde, Kumar V. S. Nemmani, Nitin J. Deshmukh, Amol Dixit, Manoja K. Brahma, Umakant Bahirat, Lalit Doshi, Rajiv Sharma, H. Sivaramakrishnan [1]
Molecular Tour
The paper reports a novel scaffold containing isocytosine moiety that was discovered for xanthine oxidase inhibition by virtual screening and enzymatic assay. Several co-crystal structures of Xanthine oxidase and xanthine dehydrogenase (which do not differ in conformation of active site) were studied to understand key interactions for enzyme inhibition. Docking of novel hits and inactive compounds was performed (to protein in PDB id : 1VDV) to understand the ligand-protein interactions and hence, for structure-based design of more potent molecules. The paper reports the directions for modification of the hit compound derived from these considerations, which are reported below. The mechanism of action of the novel hits are like piraxostat and febuxostat (pure inhibitors”) and not like allopurinol & FYX-051 (“substrate inhibitors”).
Introduction to functional aspect of xanthine oxidase :
Xanthine oxidoreductase (XOR), is an oxidoreductive enzyme that is synthesized as xanthine dehydrogenase (XDH) and can be converted reversibly or irreversibly to xanthine oxidase (XO) form. It catalyzes the transformation of physiological substrates such as hypoxanthine to xanthine and xanthine to uric acid which is excreted by kidneys.1 The reaction occurs at the cofactor molybdopterin (Mo-Pt) center from where the electrons are transferred via two Fe2S2 clusters to FAD, which then passes them on to the second substrate NAD+ in case of XDH or to molecular oxygen in XO leading to the formation superoxide anion and H2O2. Excessive production and/or inadequate excretion of uric acid results in hyperuricemia is associated with conditions like gout, cardiovascular mortality and metabolic syndrome including hyperinsulinemia and hypertriglyceridemia. Alleviating hyperuricemia, therefore, has therapeutic significance, and XO is a key target towards this end.
Important interactions of XO inhibitors with protein active site :
Piraxostat (PDB code 1VDV- Fig2A)5 and Febuxostat (PDB code 1N5X)3, show several interactions with the active site residues of the protein. The carboxyl group is involved in electrostatic interactions with guanidinium group of Arg880 and H-bonds to Thr1010 as well. The ring nitrogen is involved in H-bond interaction with Glu802. Asn768 forms another crucial H-bond with the cyano group of the ligand. Besides these polar interactions, a number of hydrophobic interactions are observed as well. The heteroaromatic ring is pi-stacked between Phe914 and Phe1009. The phenyl ring has hydrophobic interactions with Leu873, Val1011 and Leu1014. The alkoxy side chain extends towards the solvent accessible region and is engaged in hydrophobic interactions with various residues at the entrance of the pocket such as Leu648, Phe649 and Phe1013.
Similar interactions have been observed by docking our isocytosine series of compounds. The pyrimidine ring pi-stacks between Phe914 and Phe1009. Highly polar groups such as –OH on pyrimidine ring correspond to carboxylate of piraxostat and retain H-bonds with Arg880 and Thr1010. The –NH2 group in the same ring H-bonds to Glu802, which seems to play the role of anchoring the molecule in appropriate pose in the active site. The methoxy group shows a few of the several hydrophobic interactions observed for piraxostat and febuxostat.
Mechanism of action of xanthine oxidase:
Currently approved drugs for xanthine oxidase inhibition are allopurinol and febuxostat. Although both bind to the xanthine-binding site of XO, they work by different molecular mechanisms of action. Allopurinol acts as a substrate that is metabolized via hydroxylation to oxypurinol by Mo-Pt in the active site. Oxypurinol further inhibits the binding of xanthine by co-ordinating with Mo-Pt.2 Febuxostat binds tightly in the active site and blocks the binding of xanthine, without interacting with Mo-Pt.3 FYX-051 or topiroxostat currently in Phase II clinical trials also interacts with Mo-Pt just like allopurinol whereas piraxostat is akin to febuxostat.4
The mechanism of metabolism of substrate by XO requires that an electrophilic carbon next to a ring nitrogen of the substrate be positioned adjacent to Mo-Pt, with nitrogen towards Glu1261. Glu1261 acts as a general base and abstracts a proton from Mo-Pt hydroxyl group. The ionized Mo-Pt facilitates nucleophilic attack on the electrophilic carbon center. This type of motif is seen in the substrate inhibitors, allopurinol and FYX-051.4 Febuxostat and piraxostat do not possess this motif and do not get metabolized by Mo-Pt.
Our hit has a novel isocytosine scaffold that has a nitrogen in the desired position, but the carbon is substituted with –NH2, and is not available for attack by Mo-Pt. Hence our compounds are "pure inhibitors" and not "substrate inhibitors".
References
1. Pauff, J. M.; Cao, H.; Hille, R. J. Biol. Chem. 2009, 284, 8760.
2. Truglio, J. J.; Theis, K.; Leimkuhler, S.; Rappa, R.; Rajagopalan, K. V.; Kisker, C. Structure. 2002, 10, 115.
3. Okamoto, K.; Eger, B. T.; Nishino, T.; Kondo, S.; Pai, E. F.; Nishino, T. J. Biol. Chem. 2003, 278, 1848.
4. Okamoto, K.; Matsumoto, K.; Hille, R.; Eger, B. T.; Pai, E. F.; Nishino, T. Proc. Natl. Acad. Sci. U S A. 2004, 101, 7931.
5. Fukunari, A.; Okamoto, K.; Nishino, T.; Eger, B. T.; Pai, E. F.; Kamezawa, M.; Yamada, I.; Kato, N. J. Pharmacol. Exp. Ther. 2004, 311, 519.
