The compounds were ranked based
The compounds were ranked based on the Glide XP Score, as shown in . All 15 compounds were subject to a radiometric assay previously used in our lab to evaluate their ability to inhibit DHFR steady-state activity in recombinant TS-DHFR (A). Compound demonstrated greater than 50% inhibition of DHFR at 500 μM (, ). In a separate experiment we evaluated the inhibition of ache inhibitor against TS activity and observed little inhibition of the TS reaction (data not shown), which is not surprising given the large distance separating the proposed non-active site pocket from the TS domain. Although poor solubility at concentrations beyond 500 μM precluded determination of an IC value, it is clear from the radiometric experiments that compound exhibits dose-dependent inhibition of DHFR (). Two additional compounds, and , also inhibited DHFR at 500 μM by 32.6% and 21% respectively, while the remaining compounds were not effective inhibitors (A, ). In order to determine the mode of inhibition for compound , we conducted a steady-state rate profile of DHFR with its substrate, dihydrofolate, at varying concentrations of compound using the radiometric assay. In theory, noncompetitive inhibition would suggest the binding of compound to an allosteric binding site unique from the catalytic binding site of dihydrofolate. Indeed, compound displayed noncompetitive (mixed) inhibition for dihydrofolate at 0, 100, and 250 μM of the compound, as observed by a concentration-dependent decrease in , 2.17 ± 0.04 s, 1.90 ± 0.05 s, and 1.20 ± 0.04 s, respectively, while the change in was insignificant at 1.2 ± 0.2 μM, 1.3 ± 0.3 μM and 1.4 ± 0.4 μM, respectively (A and 3B). One possible explanation for the noncompetitive mechanism observed is that compound is displacing NADPH, the cofactor in the DHFR-catalyzed reduction of dihydrofolate to tetrahydrofolate. If this is the case, then the activity of compound is mediated by binding to the DHFR active site and not the proposed binding pocket in C. In order to rule out binding to the active site, we repeated the steady-state rate profile of DHFR at varying concentrations of compound and NADPH, this time keeping the dihydrofolate concentration constant. Due to the limitations of our assay, we were only able to determine the observed rate with respect to 100 µM NADPH. Compound appears to display noncompetitive inhibition with respect to NADPH, which is evident by a decrease in , 1.98 ± 0.09 s, 1.60 ± 0.06 s, and 1.33 ± 0.08 s at 0, 100, and 250 μM of compound , respectively. Upon closer inspection of subsequent docking models of compound with TS-DHFR, we observed two types of predicted binding configurations between the compound and the non-active site pocket. In the first configuration, the 1-(4-bromo-2-methylphenyl)-3-phenylthiourea moiety of compound is directed into the pocket (A), while the 2-hydroxy--phenylbenzamide moiety is embedded in the pocket in the second configuration (B). Furthermore, each configuration forms unique contacts with residues in the non-active site pocket. We evaluated these observations by conducting a structure activity relationship (SAR) study utilizing commercially available compounds derived from the structures of the two different embedded moieties of compound At 500 µM of compound, derivatives of the 1-(4-bromo-2-methylphenyl)-3-phenylthiourea moiety produced no greater than 30% inhibition of DHFR (data not shown). On the other hand, several derivatives of the 2-hydroxy--phenylbenzamide moiety demonstrated significant inhibitory activity (). In particular, we observed that compounds containing halogen substituents para to the hydroxyl moiety of the phenolic ring (A, red circle) demonstrated greater inhibitory activity against DHFR. Moreover, compounds containing substituents larger than a methyl group meta to the hydroxyl moiety of the phenolic ring (A, blue circle) displayed little DHFR inhibition. Finally, we observed that compounds containing chlorine substituents meta to the amide linker of the second aromatic ring (A, green circles) displayed greater levels of inhibition against DHFR, while most other substituents to this ring resulted in compounds with little or no inhibitory activity (A, ). These observations are consistent with the structure of compounds , , , , and (B), which, at 500 μM, inhibited DHFR by 49%, 61%, 61%, 54%, and 47%, respectively. Due to poor solubility at concentrations greater than 500 µM, we were not able to determine IC values for compounds , , , , and however these compounds do exhibit similar dose-dependent inhibition of DHFR as observed for the parent compound ().