A few purine and pyrazolo pyrimidine-based compounds were screened for inhibitory property against XOD-catalyzed hydroxylation using xanthine and 6MP as substrates (data not shown). 10 μM of either of the substrates and 2.8 U/ml of XOD were taken for the IC₅₀ determination (Table 1). IC₅₀ values of allopurinol for the enzymatic conversion of xanthine to uric acid and 6MP to 6TUA are found to be almost same (2.36 ± 0.03 and 1.92 ± 0.03 μM) and therefore, suggest that allopurinol is equally efficient in inhibiting enzymatic conversion of both xanthine to uric acid and 6MP to 6TUA formation. Since allopurinol is a suicide inhibitor of XOD and the inhibition develops time-dependently 31, so the IC₅₀ value of allopurinol was calculated for the late phase as well by carrying out the reaction for 60 min and the IC₅₀ values of allopurinol for XOD corresponding to the substrates hypoxanthine and xanthine were 0.55 ± 0.45 and 1.65 ± 0.25 μM, respectively. While for 6MP, the IC₅₀ values were already calculated from 60 min reaction. Among many compounds tested, two purine-based compounds namely, AHMP and APT demonstrated a lower IC₅₀ value for the 6MP to 6TUA reaction (0.54 ± 0.01 μM and 2.57 ± 0.08 μM, respectively), although showed a reasonably higher IC₅₀ value for the xanthine to uric acid formation reaction (17.71 ± 0.29 μM and 16.38 ± 0.21 μM, respectively). The lower IC₅₀ values for 6MP hydroxylation than xanthine indicate that AHMP and APT inhibit hydroxylation of 6MP and xanthine with variable efficiencies. These two purine-based compounds are found to be showing no such difference in the IC₅₀ value in the first and the late phases and none were found to be XOD substrates. However, like allopurinol, the recently discovered XOD inhibitors, 6-(N-benzoylamino)purine 29 and also 4-aminopyrazolo 3, 4-d pyrimidine (APP) 30 did not exhibit any preferential inhibition of the 6MP hydroxylation over xanthine. The results imply that the structure of the inhibitor plays a major role in regulating this preferential inhibition mechanism.

We also monitored the substrate-induced enzymatic electron transfer to the acceptors like 2,6-dichlorophenol indophenol (DCPIP) and cytochrome c and found that these two preferential inhibitors did not release either any electrons or generate any ROS on interaction with XOD while allopurinol led to substrate-induced enzymatic electron transfer via DCPIP (data not shown) 2930. Some researchers report the generation of superoxides from allopurinol in large amounts 23.

In order to determine the extent of enzymatic hydroxylation of xanthine and 6MP into their corresponding products when the two substrates were present together in the same reaction mixture, we carried out a bi-substrate simulative experiment.

Percent residual activity for generation of uric acid and 6TUA was determined in the presence of various concentrations of AHMP, APT and allopurinol (Table 2). XOD-mediated hydroxylation reaction of xanthine and 6MP (bi-substrate) led to generation of uric acid and 6TUA with progress of time, as shown in Figure 3. Absorption spectra of the time dependent XOD-mediated bi-substrate reaction in the presence of APT and AHMP are depicted in Figure 4 and Figure 5, respectively. Almost complete inhibition of 6MP hydroxylation while little inhibition of the xanthine hydroxylation reaction was achieved in the presence of 5 μM APT and 2 μM AHMP (Here, variable inhibitor concentrations were chosen depending on different IC₅₀ values for the two substrates). Accounting for the percentage inhibition using these inhibitors, we found that using 2.5 μM allopurinol, the uric acid and 6TUA forming residual enzyme activity was 30% and 16%, respectively. Using 2.5 μM APT, the residual enzyme activity for xanthine and 6MP hydroxylation was reduced to 80% and 25%, respectively and while using 2.5 μM AHMP it resulted in residual 64% uric acid formation activity and 18% 6TUA formation.

Therefore, the results suggest that when the two substrates, 6MP and xanthine are present together along with either of the preferential inhibitors, the conversion of xanthine to uric acid is more favored than 6MP to 6TUA conversion. The results also suggest that a critical concentration of the inhibitor could be used to effectively inhibit the enzymatic conversion of 6MP to 6TUA in the branched pathway. A similar invitro tri-substrate simulative studies was carried out with three substrates of XOD namely, hypoxanthine, xanthine and 6MP in the absence and presence of preferential inhibitors. Identical results corresponding to the preferential inhibitors were obtained indicating the discriminatory inhibition of the 6MP to 6TUA conversion and not hypoxanthine to uric acid conversion by the action of AHMP and APT (data not shown).

To study mechanism of inhibition and distinguish the interaction of XOD with the substrates, xanthine and 6MP and inhibitors, AHMP and APT; we studied the steady-state kinetics of XOD with respect to the substrates and inhibitors. The double reciprocal Lineweaver Burk (LB) plots were drawn to find the mechanism of inhibition of XOD by APT and AHMP (Figure 6, 7, 8, 9). The K_m, K_i¹, K_I¹, K_i², K_I² and k_cat values, and inhibitory mechanism exhibited by APT and AHMP are all depicted in Table 3.

Figure 6 depicts the LB plot of the steady-state inhibition with increasing APT concentrations, which show straight lines meeting at a point on Y-axis. Mechanistically, this type of inhibition is interpreted as competitive. Structurally speaking, we observe that the 8^th position in both xanthine and APT is free for hydroxylation facing the Mo site of XOD. Also, both APT and xanthine has substitution at the same positions, i.e. 2^nd and 6^th. Therefore, it seems that xanthine and APT compete for XOD active site. Consequently, it becomes evident that APT will bind to XOD at its substrate binding site. The K_i¹ value estimated from the LB plot using the equation (1) [mentioned in Methods section] were equivalent to 6.6 ± 0.28 μM with respect to xanthine (Table 3).

Figure 7 projects a LB plot of steady-state inhibition of XOD using 6MP as substrate with increasing APT concentrations, suggesting a conventional competitive inhibition mechanism. Structural explanations illustrate that 8^th position is open for action in both 6MP and APT in front of the Mo site of XOD. The K_i¹ value determined from the LB plot using the equation (1) [mentioned in Methods section] was found to be 1.30 ± 0.09 μM (Table 3).

AHMP also showed signs of efficiently inhibiting the XOD-catalyzed 6MP catabolic reaction than the xanthine hydroxylation reaction. Figure 8 demonstrates the LB plot of the steady-state inhibition of XOD using xanthine as substrate with increasing AHMP concentrations, illustrating the mixed type of inhibition like known XOD inhibitors, BOF-4272, Pd⁺², Y-700, Morin and Galagin, 32333435. The K_i² and K_I² values of 5.78 ± 0.48 μM (competitive) and 6.24 ± 0.04 μM (non-competitive) were calculated from the LB plot using the equation (4) and (5) [mentioned in Methods section]. Its non-competitive inhibition property can be explained on the basis that 8^th position is occupied by SH group in AHMP, which is found free in xanthine. Besides, the presence of NH₂ group at 2^nd position in AHMP in place of an OH group in xanthine makes it faultless contender to bind at any site other than active site on XOD. And its competitive inhibition property may be due to the presence of OH group present at the 6^th position in both AHMP and xanthine and thus, they compete for the enzyme active site.

For XOD-catalyzed 6MP catabolic reaction, AHMP shows evidence of a mixed inhibition mechanism (Figure 9) indicating that native XOD is capable of forming two inhibitory complexes with AHMP. Occurrence of SH group at positions 6^th in 6MP and at 8^th in AHMP makes the latter an ideal applicant to compete with 6MP for active site and consequent inhibition of XOD. However, the existence of extra groups (OH and NH₂) in AHMP at two positions makes it attach at a site other than the active site of the enzyme. K_i² and K_I² values for inhibition were calculated out to be 0.96 ± 0.01 μM (competitive) and 0.98 ± 0.06 μM (non-competitive) using the equations (4) and (5) [mentioned in Methods section]. The values of the two inhibition constants of AHMP do not differ much, indicating that the nature of the coordination of the AHMP for the competitive and non-competitive binding sites is similar.

Single dose acute toxicity was evaluated for AHMP and APT and compared with allopurinol, the standard drug used in the present investigation. No differences in general behavioral toxicity and no mortality were observed after the administration of the compounds. No significant changes in the initial and final body weight were observed for any of the compounds. The weekly food intake decreased for the groups treated with AHMP, APT and allopurinol as compared to vehicle treated groups. However, AHMP and APT treated groups food intake was marginally less as compared to allopurinol treated groups. Gross necropsy revealed no changes in vital organs. No difference in organ weight was observed as compared with the control groups.

Mixed-type inhibition includes competitive and non-competitive inhibition. Competitive inhibition is exhibited when the substrate and the inhibitor compete for the substrate binding active site but non-competitive inhibition is exhibited when the inhibitor binds at a site other than the active site. Following the crystallographic studies performed on XOD, no peripheral binding site has been reported in XOD catalytic subunits 3637. For the past 50 years, it was assumed that the two XOD subunits carry out catalysis independently but a recent report 38 on the cooperative binding of the two subunits of XOD may support the non-competitive inhibition mechanism exhibited by AHMP. AHMP possibly interacts with the XOD domains distal to the substrate binding site also other than the substrate binding site, thereby possibly resulting in allosteric effects that attenuate the enzymatic activity.

Since XOD acts via a ping-pong mechanism, alternating between the oxidized and reduced forms 39, so another plausible explanation for the mixed inhibition exhibited by AHMP is that AHMP binds to both the oxidized and reduced form of XOD. Allopurinol (a known weak competitive inhibitor) and nitric oxide are known to strongly bind to the reduced state of XOD 4041. APT being a competitive inhibitor thus, might not be binding to the oxidized state but to the reduced state of XOD. The difference in the mechanism of inhibition exhibited by AHMP and APT must be possible due to the structural dissimilarities between the two inhibitors.

The K_m, k_catand K_i values of XOD corresponding to the substrates, 6MP and xanthine and the inhibitors, APT and AHMP behave as the factors indicating the type of their binding and catalytic interactions with the enzyme. In the biochemical reaction,

where, E is enzyme, S is substrate, P is product, k₁ and k₃ are the rate constants for the forward reactions and k₂ is the rate constant for the backward reaction.

By definition, K_m of any biochemical reaction can be mathematically expressed in terms of rate constant 42 as

K_m = (k₂+ k₃)/k₁

Lower K_m implies a lower dissociation of ES into E+S or E+P while higher K_m implies higher dissociation of ES into E+S or E+P. While looking at the turnover number (k_cat) of xanthine and 6MP, we found that it is the dissociation of the corresponding ES complexes into E+P and not E+S.

The inhibition constant K_i on the other hand can be defined 42 as:

where, I is inhibitor, k₁ is the rate constant for the forward reaction and k₂ is the rate constant for the backward reaction

K_i = k₂/k₁

Lower K_i implies lower dissociation of EI complex into E + I and higher association of E and I, while higher K_i means higher dissociation of EI complex and lower association of E and I.

K_m and k_cat values of XOD using 6MP as substrate implied that the conversion of XOD-6MP (ES) complex into its product 6TUA is slower as its turnover number is much lower (2.85 ± 0.02 μM/min) than the conversion of XOD-xanthine complex into its corresponding product (Table 3). Additionally, XOD-6MP (ES) complex is also less favored as the K_m in this case is high (6.01 ± 0.03 μM). Considering xanthine as substrate of XOD however, the situation is different. The high k_cat (114.54 ± 0.24 μM/min) indicates that the forward reaction of xanthine-XOD into uric acid (ES into E+P) is faster as compared to its dissociation into other counterpart (E+S) and lower K_m (2.65 ± 0.02 μM) indicates that xanthine-XOD (ES) complex formation is also more favored. Therefore, the higher turnover of xanthine to uric acid conversion in comparison to 6MP to 6TUA conversion is favored only if the ES complex formation is favored rather than its dissociation into E+S which does not occur in case of 6MP-XOD interaction.

Now, we can presume a correlationship of K_m and K_i of XOD with respect to xanthine and 6MP, respectively. When 6MP and APT (or AHMP) interact with XOD, lower K_i implies stronger XOD-AHMP (EI) complex formation and AHMP will more efficiently dissociate 6MP from XOD active site while higher K_m implies less favored XOD-6MP (ES) complex formation and subsequently, slower 6TUA formation, further supporting that XOD and 6MP (E+S) formation is much preferred in 6MP as compared to xanthine. However, when xanthine and APT (or AHMP) interact with XOD, higher K_i implies weaker and slower rate of formation of XOD-AHMP (EI) complex and the inhibitor AHMP is not efficient in displacing xanthine from XOD and thus, the enzymatic hydroxylation of xanthine continues while lower K_m implies more favored formation of XOD-xanthine (ES) complex and subsequently, higher formation of uric acid.

It will be right to say that these preferential inhibitors, AHMP and APT displace 6MP efficiently from weak XOD-6MP complex while AHMP and APT are incapable of displacing xanthine from strong xanthine-XOD complex (Figures 10 and 11). In other words, substrate hit and inhibitor displacement takes place from XOD active or non-active site for xanthine-XOD reaction but inhibitor hit and substrate displacement for 6MP-XOD reaction. It can be stated that the 6MP to 6TUA hydroxylation is discriminately inhibited in contrast to xanthine to uric acid hydroxylation by these preferentially inhibitors.

In order to evaluate the toxicity of the preferential inhibitors of XOD, the single dose acute toxicity study was performed for both the preferential inhibitors on swiss male mice following a fourteen days observation period. The body weight of the animal ranged from 25–30 gm. Three different doses (2.5, 5, 10 mg/kg) for the two compounds were selected, using allopurinol as the standard drug and administered intravenously 55. Parameters, e.g., body weight, food intake, mortality, general behavioral toxicity were observed after the administration of the compounds for fourteen days. Gross necropsy of each animal of all groups was conducted and organ weight was recorded after the termination of the study.