Summary: Heme Binding Studies

2.3. Mechanistic studies: Heme binding studies
Chloroquine and other quinoline derivatives are believed to show their antimalarial activity by inhibition of hemozoin formation within the parasite food vacuole [49]. Hemozoin was originally considered to be formed by the polymerization of heme [50], but it has now been demonstrated that it is a crystalline cyclic dimer of ferriprotoporphyrin IX [51]. It is widely accepted that CQ accumulates in the plasmodium food vacuole and binds to some form of parasite heme/hematin, and inhibits hematin polymerization [52-55]. This is a non-enzymatic process in which hematin monomer (heme) released from parasite hemoglobin digestion is converted into hemozoin, also known as malaria pigment. Hemozoin is an

Docking studies
In the present study, the binding mode for novel 4-aminoquinoline-pyrimidine based molecular hybrids in the active site of wild type PfDHFR-TS and quadruple mutant PfDHFR-TS (N51I, C59R, S108 N, I164L) protein structures was explored using molecular docking studies. For this purpose, the prepared 3D structures of the compounds were docked in the binding pocket of both the wild type PfDHFR-TS and quadruple mutant PfDHFR-TS (N51I, C59R, S108 N, I164L) structures.
The active aminoquinoline-pyrimidine conjugates interacted with wild and mutant PfDHFR-TS forming H-bond and π-π interactions. Table 4 shows the results of docking for the active compounds. The Glide XP Gscores and glide energies clearly indicate that the most active compounds in the study exhibited significant binding affinities towards the wild (Glide energy range -58.11 kcalmol-1 to -44.32 kcalmol-1) and quadruple mutant (Glide energy range -56.14 kcalmol-1 to -43.74 kcalmol-1) PfDHFR-TS structures and the energy ranges are comparable to that of reference compounds (pyrimethamine, cycloguanil and WR99210) and the native substrate of DHFR dihydrofolate (Table 4).
<Insert Table 4

The in vitro evaluation of these hybrids against D6 and W2 strains of P. falciparum depicted activity in the micromolar range. Also, these hybrids exhibited high selectivity indices and low toxicity against the tested cell lines. Three compounds (7g, 8a and 8b) exhibited better antimalarial activity (IC50 = 0.019 µM to 0.033 µM) than the standard drug chloroquine (IC50 = 0.035 µM) against CQ-sensitive strain, whereas except 7a, 7b, 8c, 9a, 9b and 9e, all the compounds showed better activity than chloroquine against CQ-resistant strain. Some of the compounds showed resistance index value close to one which indicates that the antimalarial activity of these molecular hybrids are comparable between the CQ-sensitive and CQ-resistant strains of P. falciparum and hence suggesting that the CQ-resistance mechanism has little effect on the antimalarial activity of these compounds. Interestingly, all the compounds displayed better activity than pyrimethamine (>19 µM) against the CQ-resistant strain W2. Further, the binding capability of compound 7c was evaluated with heme to find out the probable mode of action of these hybrids. The molecular docking studies of active compounds from the in vitro studies were performed and all the compounds showed good interaction with the binding sites of PfDHFR, comparable to the inhibitors and substrates. The calculated ADMET