The previous study of Jacoby et al.
) on a weakly diffracting monoclinic crystal form of T. cruzi
TryR soaked in a quinacrine solution was limited by the poor quality of the diffraction data. Of the four active sites present in the asymmetric unit, the resulting model had only one site occupied by the ligand and this at occupancy of 0.3. These data are not publicly available, but coordinates were kindly provided by the authors for comparative purposes. Their analysis only identified a single inhibitor molecule bound near the hydrophobic pocket mainly formed by the Trp22
residues. The acridine was modelled in a position perpendicular to and overlapping with Q1
in our structure, with the ring nitrogen and chlorine substituent pointing toward the side chain of Trp22
. The considerable differences observed between the two structures may be a consequence of the different inhibitor molecules, with the QM capable of covalent modification of the protein.
However, we note that a previous kinetic analysis of several acridine derivatives indicated that more than one molecule of these inhibitors bind per TryR monomer (19
). Unfortunately these data did not allow determination of the positions or number of interaction sites and it was assumed that one inhibitor bound in the active site in the fashion suggested in the previous model of a quinacrine complex. A second inhibitor was speculated to bind in a cavity at the dimer interface or to a hydrophobic patch at the edge of the active site. Moreover, multiple interaction sites of tricyclic molecules have been identified in a previous study that used photoaffinity labelling to examine the interaction of the tricyclic drug fluphenazine with TryR (40
). These experiments indicated multiple binding sites for tricyclic molecules in the TryR active site, yielding fluphenazine / TryR monomer molar ratios in the range of 2-5. This result was interpreted in light of the known ability of phenothiazine drugs to stack in solution, a property that they share with quinacrine (39
). Our data extend these findings by showing that the two molecules of QM which react to inactivate TryR actually bind in the active site, with no evidence for interaction elsewhere on the protein.
The observation that one of the chloroethyl groups on Q2 is unreacted in the native protein offers an explanation for two apparent discrepancies in our biochemical and mass spectrometry studies. First, the small amount of cross-linked dimer observed only in the EH2 form of TryR could arise during denaturation of the protein for SDS-PAGE. Second, digestion with trypsin would allow the mono-alkylated peptide fragment to cross-link with other peptides in the mixture, accounting for our failure to observe the predicted products by mass spectrometry.
The structure of this QM-TryR adduct represents an end-point of an alkylation reaction and might not therefore represent the actual sites for the non-covalent quinacrine interaction. However, several lines of kinetic evidence presented above indicate that the positions in which the QM are found in the crystal structure represent the actual sites at which non-reactive acridine molecules interact with TryR. Notably, the observation that a reversible EI complex (Ki
36 μM) is formed between QM and the EH2
form of TryR indicates that the inhibitor does bind to the active site region. Further support for this conclusion is provided by the protection afforded by the T[S]2
mimic, Mel T. Since it is probable that the binding mode of the arsenic-T[SH]2
conjugate Mel T is similar to that of the natural T[S]2
substrate, the ability of this inhibitor to protect against QM inactivation can be interpreted using the structure of the TryR-T[S]2
). In this structure both the Q1
binding sites are occupied by the spermidine component of the T[S]2
Unfortunately, the large size of the TryR substrate-binding cleft means that many different orientations of small ligands are possible. However, the synergistic effect of quinacrine on QM inactivation at low concentrations and the antagonistic effect at high concentrations clearly indicate that the two regions of the active site which bind these inhibitors can interact. This interaction appears to depend upon the planar tricyclic ring system since the competitive inhibitor clomipramine, which possesses a non-planar ring, obeys simple one-site competitive kinetics and protects TryR against QM inactivation. These data are therefore entirely consistent with the observed QM binding sites, which are adjacent to each other and allow stacking of the acridine ring systems.
Based on the structure of the alkylated enzyme and our kinetic data we propose a model to explain the different effects of quinacrine and clomipramine on the QM inactivation reaction (). Since inactivation of the EH2 enzyme proceeds rapidly through the reaction of a mustard group with the Cys53, occupation of the Q2 binding site by QM will be required for this reaction to occur. However, the majority of the contacts made by the QM moiety in this position are through van der Waals’ interactions with the acridine ring of the Q1 ligand. This implies that the affinity of site Q2 will be enhanced by an increase in the occupancy of site Q1. Based on the inactivation kinetics of QM with the oxidised enzyme, the affinity of this site is low (Ki > 40 μM). Hence, addition of quinacrine to a QM inactivation reaction will titrate site Q1 and may allow more quinacrine and QM to bind to site Q2, thus producing an increase in the overall rate of inactivation. Since Cys53 is only available for alkylation in the EH2 form, enhanced inactivation is not observed in the E form. At higher concentrations of quinacrine, QM will be increasingly excluded from both sites, resulting in decreased inactivation. In contrast, the occupation of site Q1 by clomipramine would be predicted to simultaneously reduce the occupancy of site Q2 and thus reduce the rate of inactivation, since the non-planar ring of this inhibitor would not allow π-stacking interactions.
Kinetic and structural models of the effects of clomipramine and quinacrine on inactivation of trypanothione reductase by quinacrine mustard
The description of two interacting binding sites in the active site of TryR has important implications for drug design and may allow the design of far more potent inhibitory molecules than can be produced from the simple tricyclic scaffold. This could be achieved through the design of molecules that could bind simultaneously to both of these sites. Indeed, previous studies may have unwittingly exploited this approach; for example, the finding that bis-substituted polyamine derivatives are significantly better TryR inhibitors than their mono-substituted equivalents (41
). Importantly, these polyamine derivatives include some of the most potent TryR inhibitors which have yet been described, with N1
-bis-(5-bromo-3-indole acetyl)spermine having a Ki
of just 76 nM against T. cruzi
). However, the flexibility of these spermine and spermidine derivatives could produce an entropic penalty upon binding. The identification of adjacent tricyclic binding surfaces may therefore allow the construction of conformationally constrained inhibitors with affinity for both sites, resulting in even tighter binding.
Although TryR provides an attractive target for selective inhibition, purely competitive inhibitors of this enzyme may not be ideal as drug candidates, unless of low nanomolar affinity. Since there are extremely high concentrations of T[SH]2
in the parasite cell (reaching 5 mM in L. donovani
)), inhibition of TryR could result in the accumulation of sufficient T[S]2
to partially reverse the effects of a competitive inhibitor (44
). Indeed, more than 90% inhibition of TryR is required to kill T. brucei
) and L. donovani
can survive an 85% reduction in TryR activity with no effect upon either their viability or their ability to metabolise H2
). Collectively, these data suggest that the ideal TryR inhibitor would be one that showed either tight-binding or irreversible inhibitory activities. Although the mustard groups render QM too reactive to be considered as a lead compound, the detailed mechanistic and structural data provided in this study may allow the design of less toxic compounds with similar activities.