Common side effects of allopurinol include: Rash.

sh. This is a common side effect, but it can be a sign of a very serious or lifethreatening condition. Diarrhea and nausea. Increased gout attacks at first. To avoid this, your doctor may also prescribe either colchicine, which blocks the inflammation caused by uric acid crystals, or low-dose nonsteroidal anti-inflammatory drugs (NSAIDs ). After normal uric acid levels have been maintained for 6 to 12 months and no further attacks occur, colchicine or NSAIDs do not need to be taken. Common side effects of febuxostat include: Abnormal liver function. Dizziness.

Xanthine oxidase inhibitors are of two kinds: purine analogues and others. Purine analogues [2] [3] include allopurinol, oxypurinol, and tisopurine. Others include febuxostat and inositols (phytic acidand myo-inositol).

Functional aspects 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 [2] xanthine andxanthine to uric acid which is excreted by kidneys. 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 (PDB [3] code 2w3s) or to molecular oxygen in XO leading to the formation of superoxide anion and H 2O2. Excessive production and/or inadequate excretion of uric acid results in hyperuricemia and 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) and febuxostat (PDB code 1n5x) , show several interactions with the active site residues of the protein. The carboxyl group of piraxostat 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. The cyano group of the ligand forms another crucial Hbond with Asn768. 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. Piraxostat is in green, Mo-Pt is in
[4] [5]

deep-sky-blue, residues are colored according to the type of interaction with ligand salmon for pistack, magenta for other hydrophobic and cyan for polar interactions. Similar interactions have been observed by docking our isocytosine series of compounds. The pyrimidine ring pi-stacks between Phe914 and Phe1009 (compound 1 is shown). 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. Similarities and differencesin the interactions of Compound 1 and piraxostat can help in structure-based design to improve activity of isocytosine series. 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 [6] site. Oxypurinol further inhibits the binding of xanthine by co-ordinating with Mo-Pt. Febuxostat binds [5] tightly in the active site and blocks the binding of xanthine, without interacting with Mo-Pt. FYX-051 or topiroxostat currently in Phase II clinical trials also interacts with Mo-Pt just like allopurinol whereas [7] piraxostat is akin to febuxostat. 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, [7] allopurinol and FYX-051. 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".

Xanthine oxidase (XO or XOA) is an enzyme that catalyzes the chain reactions of hypoxanthine oxidizing to xanthine and xanthine oxidizing to uric acid and hydrogen peroxide (H2O2). Oxidation requires the addition of oxygen and water. This process is important because it explains how humans are able to metabolize nitrogen compounds called purines. When xanthine oxidase, a type of xanthine oxidoreductase (XOR), undergoes a reversible process called sulfhydryl oxidation, it becomes converted to xanthine dehydrogenase. In sulfhydryl oxidation, a sulfur-containing organic compound is used rather than water. Xanthine dehydrogenase is also able to catalyze the oxidation of purines. The substrates for this enzyme include xanthine, nicotinamide adenine dinucleotide and water.

Metabolism OF PURINES

Uric acid is the end product of purine catabolism. - Adenosine is first deaminated to inosine by adenosine deaminase, then phosphorolysis of N-glycosidic bonds of inosine and guanosine, catalyzed by purine jnucleoside phosphorylase release ribose 1-phosphate and a purine base. - Hypoxanthine and guanine next form xanthine in reactions catalyze by xanthine oxidase and guanase, respectively. - Xanthine is then oxidized to uric acid in a reaction catalyzed by xanthine oxidase.
PROTEIN STRUCTURE DEHYDROGENASE
Crystal structure of the xanthine dehydrogenase dimer divided into the three major domains and two connecting loops. The two monomers have symmetry related domains in the same colors, in lighter shades for the monomer on the left and in darker shades for the monomer on the right. From the N to the C terminus, the domains are the iron/sulfur-center domain (residues 3-165; red), the FAD domain (residues 226-531; green), and the molybdopterin center (Mo-pt) domain (residues 590-1331; blue). The loop connecting the iron/sulfur domain with the FAD domain (residues 192-225) is shown in yellow, the one connecting the FAD domain with the Mo-pt domain (residues 537-589) is in brown, and the N and C termini are labeled. The FAD cofactor, the two iron/sulfur centers, the molybdopterin cofactor, and the salicylate also are included OXIDASE
Crystallographic structure (monomer) of bovine xanthine oxidase. The bounded FAD (red), FeS-cluster (orange), the molybdopterin cofactor with molybdenum (yellow) and salicylate (blue) are indicated.

ALLOWING SMALLER OXYGEN MOLECULES TO ACCEPT ELECTRONS FROM FAD

CATALYTIC MECHANISM
XDH can be the target of oxidation and proteolysis, modifications that change the binding specificity of the enzyme and lead to the formation of XO. Both XDH and XO catalyze the terminal steps in the metabolism of purines. However, in the case of XDH, which binds NAD+, the electrons deriving from the oxidation of hypoxanthine or xanthine reduce NAD+ to NADH. In contrast, XO, being no longer able to bind NAD+, generates electrons that are transferred directly to molecular oxygen, leading to the formation of the ROS superoxide. The alteration in binding specificity for NAD+ is irreversible in the case of proteolysis but can be reversed by reducing agents in the case of oxidation. Nevertheless, it is important to point out that the dehydrogenase form of XOR can also use molecular oxygen and produce ROS, although less efficiently than the oxidase form. In addition, in certain conditions, XOR can function as an NADH oxidase, generating ROS without metabolizing xanthine or hypoxanthine. The catalysis in this case occurs at the FAD site and does not involve the molybdenum (Mo) cofactor and therefore cannot be prevented by XOR inhibitors such as allopurinol, which target the Mo cofactor (27). Moreover, XO can generate nitric oxide (NO) by catalyzing the reduction of nitrate to nitrite and nitrite to NO in the presence of NADH as electron donor. These reactions can be completely blocked by the Mo-directed XOR inhibitor, allopurinol, or the FAD-

directed inhibitor, diphenyliodonium (DPI). Therefore, nitrate reduction to nitrite as well as nitrite reduction to NO occur at the Mo center, whereas NADH is oxidized at the FAD site. Mechanism of phytic acid:
Phytic acid (known as inositol hexakisphosphate (IP6), or phytate when in salt form), discovered in [1] 1903, a saturated cyclic acid, is the principal storage form of phosphorus in many plant tissues, [2] especially bran and seeds. Phytate is not digestible to humans or nonruminant animals, so it is not a source of either inositol or phosphate if eaten directly. Moreover, phytic acid chelates and thus makes unabsorbable certain important minor minerals such as zinc and iron, and to a lesser extent, also macro minerals such as calcium and magnesium; phytin refers specifically to the calcium or magnesium salt form of phytic acid. phytic acid interferes with the formation of ADP-iron-oxygen complexes that initiate lipid peroxidation. Both phytic acid and myo-inositol inhibited XO-induced superoxide-dependent DNA damage. Mannitol inhibited the DNA strand break. Myo-inositol may act as a hydroxyl radical scavenger. The antioxidative action of phytic acid may be due to not only inhibiting XO, but also preventing formation of ADP-ironoxygen complexes.