Compounds bind to the NQO enzymes, stacking their rings between the FAD isoalloxazine ring and the plane formed by polar residues 128 and 178 from the other monomer. Tyr 128 provides polar interactions with the rings and other groups of the compounds. Apolar groups lay at a polar patch above loop L6 or contact Trp 105 and Phe 106. In NQO1, either the Nε of His 161 or the OH of Tyr 126′ provides polar interactions with one of the quinone oxygens. Larger substituents could interact with the NAD(P)+ cleft, increasing complementarity with the protein. RH1, ARH019 and E09 bind with their benzoquinone ring in one side of the pocket, stacked between Tyr 128′ and flavin rings A and B. Tyr 128′ swings over the substrate, making contacts with the aromatic core of the drugs (Fig. 15). RH1 and ARH019, a benzoquinone and an indolequinone, bind to NQO1 with a similar spatial arrangement, such that their pharmacophore atoms overlap with a low RMS deviation (≤0.6 Å). Surprisingly, this is not the case with the two indolequinones, EO9 and ARH019. Although chemically highly similar (they differ only in their substituents at position 2 and 5), they bind to the enzyme in different orientations. EO9 and ARH019 bind hNQO1 in dissimilar ways even though both are aziridinylindolequinones. The comparison of the reduction kinetics of a group of closely related compounds, studied by Bailey et al. (EO9 homologs) and by Beall et al. (ARH019 homologs), illustrate the effect of small differences in the substituents (OMe, aziridinyl, methyl-aziridinyl, open aziridinyl) at the position 5. Analogous compounds have similar relative rate. of reduction within one subfamily. Interestingly, species differences have a greater effect on EO9 than on the other prodrugs: EO9 is reduced 27 times faster by rNQO1 than by hNQO1. The binding position of EO9, closer to the center of the active site than the other compounds, apparently makes the drug more susceptible to the change in the FAD position. This compound can serve as a model for other drugs such as the antibiotic streptonigrin, another good substrate for the enzyme. Reduction rates of drugs by NQO1 similar to those described previously show a strong correlation with the modes of binding suggested by the structures. Aziridinylbenzoquinones (Fig. 12A) tolerate large substitutions only at position 3 or only at position 6. The RH1-hNQO1 complex shows that substituents at one of these two symmetrical positions points toward the outside of the pocket and the other toward the inside. Thus, only one of these positions can accommodate a large substituent. Consequently, drugs with progressively larger symmetrical substitutions have decreasing rates of reduction. In the case of indolequinones, substituents of different shapes and sizes are generally well tolerated at position 2. The indolequinone-NQO1 complexes show that substituents at this position point out of the binding pocket. A variety of small substituents at position 5 are tolerated. At position 3, small substituents have a small effect on the reduction rate, with electron-withdrawing groups increasing the rate. Indolequinones such as ES936 and ES1951 that have small substituents at 2- and 3-substituents of the type CH2R (R = leaving group) inactivate the enzyme. Reduction of these indolequinones in the catalytic site (Fig. 16) places a reactive carbonium at position 3 buried in the catalytic pocket. The resulting indolequinone iminium species is capable of alkylating nearby protein groups, inactivating the enzyme. Mass spectrometry of digested ES936-treated proteins showed that after reduction and loss of p-nitrophenol, the reactive iminium species generated alkylates one of the tyrosines in the binding site (126 of 128).
ASJC Scopus subject areas
- Molecular Biology