Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth

Considerable data now support the! hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hemain polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC-), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K-a's ranged from 0.46 to 2.9 x 10(5) M-1 compared to 4.0 x 10(5) M(-1)for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the B-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxa dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin Cc-ore dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that, electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom,is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K-a and hematin polymerization IC50 values suggests that other properties of the CQ-humatin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC50 values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that, antimalarial 4-aminoquinolines act by this mechanism.