Criterios de Evaluación para Medir la Eficacia de los REA,

The preferential binding of EtBr to reduced NQO2 is of interest because the reduced form of NQO2 appears to play an important role in cells. That is NQO2 was shown to inhibit p53 degradation in the presence of NRH, and on this basis it appears that reduced NQO2 is the “active” state of the enzyme that is capable of regulating p53 (4). To further characterize interactions between DNA intercalating agents and NQO2, the binding of EtBr, AO, and doxorubicin to both oxidized and reduced NQO2 was measured using Isothermal Titration Calorimetry (ITC). Of the three compounds, AO bound oxidized NQO2 with the highest affinity, almost 10-fold greater than both EtBr and doxorubicin (Table 5.3, Figure S5.1A-C). Preliminary titrations of AO and EtBr against reduced NQO2 indicated that their binding affinities were much higher compared to oxidized NQO2. To accurately characterize the very high affinity binding of both EtBr and AO to reduced NQO2, competition ITC was used (40). In competition ITC, a compound with high binding affinity is titrated against the target protein in complex with a relatively low affinity ligand. In the case of NQO2, chloroquine (CQ) was used as the low-affinity ligand. A direct titration of reduced NQO2 with CQ yielded a KD value of 0.57 µM

(Table 5.3, Figure S5.1G) similar to its inhibition constant (0.6 µM) determined

previously using enzyme kinetics (13). Consistent with the kinetics assays, EtBr bound most tightly to reduced NQO2, with a KD over 60 times lower compared to the oxidized

preferentially to reduced NQO2 with sub-nanomolar affinity (Table 5.3, Figure S5.1E). Doxorubicin bound to oxidized NQO2 with sub-micromolar affinity but there was no observable binding to the reduced form (Table 5.3, Figure S5.1F).

Table 5.3 Binding of NQO2 Inhibitors to Oxidized and Reduced NQO2 NQO2 Inhibitor KD (nM) ΔG (kcal/mol) ΔH (kcal/mol) -TΔS (kcal/mol) Oxidized EtBr 215 ± 33 -9.4 -7.8 ± 0.1 -1.6 AO 29.4 ± 28.7 -10.3 -7.6 ± 0.2 -2.6 Doxorubicin 274 ± 49 -8.9 -8.1 ± 0.2 -0.8 Reduced EtBra 3.47 ± 0.33 -11.5 -6.7 ± 0.1 -4.8 AOa _{0.36 ± 0.12 -12.9 -6.5 ± 0.1 -6.3} Doxorubicinb NB - - - CQ 578 ± 116 -8.5 -17.7 ± 0.5 9.2

a _{Thermodynamic parameters were determined by competition titrations against NQO2 in}

complex with CQ as described in Methods.

b _{There was no observable binding of doxorubicin to reduced NQO2 (Figure S5.1F).}

5.3.3

Crystal Structures of NQO2 with DNA Intercalating Agents

There are numerous structures of oxidized NQO2 in complex with a variety of inhibitors, but only two in which inhibitor binding to reduced NQO2 has been structurally

characterized (29, 30). In both cases, reduction of the FAD cofactor led to a striking change in the binding mode of the inhibitors. We have extended this comparative analysis by solving the high resolution crystal structures of oxidized and reduced NQO2 in

complex with EtBr and AO, as well as doxorubicin in complex oxidized NQO2 (Table S5.1). In all of the oxidized NQO2 inhibitor complexes, the inhibitors are deeply buried in the active site and sandwiched between the isoalloxazine ring of FAD and the phenyl ring of F178 (Figure 5.3A, C, and E). As such, binding of the inhibitors to oxidized NQO2 includes common aromatic stacking interactions. In the structure of reduced NQO2 with EtBr (Figure 5.3B) the overall orientation of ethidium has not changed, but it is positioned less deeply in the active site. The space vacated by the ethidium is filled with three water molecules that mediate hydrogen bonds between the amino group on the ethidium and N161 and G174 of NQO2. The situation is similar for AO: when bound to oxidized NQO2, AO is positioned deep in the active site, stacking over the oxygens of the isoalloxazine ring and making direct contact with N161 and G174 (Figure 5.3C).

However, AO has rotated and occupies a more peripheral location when bound to reduced NQO2 (Figure 5.3D), and again the space next to N161 and G174 is filled with water molecules.

The difference in binding mode for EtBr and AO resembles what was observed for chloroquine (CQ) (30) and the protein kinase CK2 inhibitor DMAT (29), which both bound to oxidized NQO2 such that they were positioned deep in the active site and made direct contact with N161 and G174. When bound to reduced NQO2, however, both CQ and DMAT were positioned in a more peripheral location, and there were water

molecules filling the space between N161, G174, and the inhibitors. It appears that the properties of the reduced isoalloxazine ring, which include a “butterfly bend” of approximately 5° (Table S5.1 and Figure S5.2) and a negative charge that will be delocalized between N1 and O2, make the region above the isoalloxazine oxygens less suitable for the aromatic stacking interactions observed in the oxidized structures. Instead, the inhibitors move away from this region which is then occupied by polar, non- aromatic water molecules.

The active site of NQO2 has a negative electrostatic potential that becomes much

stronger when the isoalloxazine ring is reduced and carries a formal negative charge (29). Both EtBr and AO are positively charged at neutral pH, which explains their preference towards the negatively charged FADH over neutral FAD. Doxorubicin also has a positive net charge, but the charge resides on non-aromatic portions of the molecule that are excluded from the active site (Figure 5.3E). Therefore, a positive charge in the planar aromatic portions of NQO2 inhibitors appears to determine their preference for reduced NQO2. This is consistent with the two other NQO2 inhibitors, chloroquine and

quinacrine, which both carry a positive charge in their aromatic portions and exhibit a marked preference for binding to reduced NQO2 (13).

NQO2 crystallizes with a dimer in the asymmetric unit, and inhibitors can sometimes adopt different orientations in the two crystallographically distinct binding sites. This was the case for both the NQO2ox-EtBr and NQO2red-EtBr structures where electron density

for a second ethidium molecule in the B-chain active site became evident during

refinement (Figure S5.3). The second ethidium interacts with the first by means of pi-pi

Figure 5.3 Binding of Ethidium, Acridine Orange, and Doxorubicin to NQO2 The inhibitors are sandwiched between the FAD co-factor (below the plane of inhibitor) and F178 (above the plane of the inhibitor) in oxidized NQO2 (A, C, E), and the

inhibitors are excluded from this region by water molecules in reduced NQO2 (B, D). (A) Ethidium is deeply buried in the active site of oxidized NQO2 making hydrogen bonds with N161 directly and to D117, T71, and G68 via two water molecules. (B) Ethidium is less buried in reduced NQO2 and makes hydrogen bonds with N161 and G174 via three water molecules on the left side of the binding site, and with G68 and T71 directly on the right side of the binding site. (C) Acridine orange binds oxidized NQO2 by making hydrogen bonds to N161 via two water molecules. (D) In the reduced structure of NQO2 in complex with AO, AO makes one hydrogen bond to E193 via a water molecule. (E) The planar moiety of doxorubicin is inserted into the left side of the binding cleft where it

is anchored by a hydrogen bond to N161. All electron density maps surrounding the inhibitors are Fc-Foomit maps generated after three rounds of simulated annealing and

contoured at 3 σ.

planar stacking of their benzyl substituents, and the rest of the ethidium is loosely sandwiched between surface residues surrounding the B-chain active site and residues from a symmetry-related molecule. We believe that this mode of binding is induced primarily by crystal packing and likely irrelevant for NQO2 in solution. In contrast to EtBr, both AO and doxorubicin bound in exactly the same manner to both subunits of the