Quinoline-triazole hybrids inhibit falcipain-2 and arrest the development of Plasmodium falciparum at the trophozoite stage

Abstract

Falcipain-2 (FP2) is a papain family cysteine protease and a key member of the hemoglobin degradation pathway, a process that is required at erythrocytic stages of Plasmodium falciparum to obtain amino acids. In this study, we report a set of 10 quinoline-triazole-based compounds (T1-T10) which exhibit a good binding affinity for FP2, inhibit its catalytic activity at micromolar concentrations and thereby arrest the parasite growth. Compounds T4 and T7 inhibited FP2 with IC₅₀ values of 16.16 μM and 25.64 μM respectively. Both the compounds T4 and T7 arrested the development of P. falciparum at the trophozoite stage with an EC₅₀ value 21.89 μM and 49.88 μM. These compounds also showed morphological and food-vacuole abnormalities like E-64, a known inhibitor of FP2. Our results thus identify the quinoline-triazole-based compounds as a probable starting point for the design of FP2 inhibitors and they should be further investigated as potential antimalarial agents.

Fig. 1. Structures of some reported FP2 inhibitors.

Fig. 2. Rationale for the design and synthesis of triazole-quinoline derivatives as FP2 inhibitors.

Fig. 3. Docking interactions of T7 and E64, a known standard FP2 inhibitor with FP2:PDB ID 3BPF; (a) binding interaction of compound T7 with FP2 showing π–π stacking between Trp43 and aromatic triazole ring (represented with red coloured dashes) and two H-bonds between the ligand and Cys39 and Asn81 of FP2 (represented with dark blue coloured dashes). (b) Showing H-bond interaction of E64 with the Gly83 and Asn173 of FP2 (represented with dark blue coloured dashes). (c) Surface view of T7 into the active site groove of the enzyme: (d) Surface pose of E64 with FP2.

Scheme 1. Synthesis of different aromatic azides. Reagents and conditions: (a) NaNO₂, HCl (3 N), ice bath (b) NaN₃, water, room temperature, 4 h, 83–90% yield.

Scheme 2. Synthesis of 7-chloro-4-(prop-2-yn-1-yloxy)quinoline. Reagents and conditions: (a) NaH, dry DMF, propargyl, alcohol room temperature, 4 h, 70% yield.

Scheme 3. Synthesis of different quinoline-triazole-based target compounds. Reagents and conditions: (a) CuSO₄·5H₂O, sodium ascorbate, (THF–H₂O, 1 : 2), 80 °C, 8 h.

Fig. 4. Recombinant FP2 protein expression and activity analysis: (A) FP2 protein was expressed in E. coli cells and purification was done with Ni²⁺-NTA column followed by refolding process. (B) Activity analysis of refolded FP2 using ZFR-AMC as substrate on dose dependent manner over a period of 30 min was measured at pH 5.5.

Fig. 5. Effect of E-64, compounds T7 and T4 on the growth and development of P. falciparum. Light microscopy images of parasitized red blood cells at different time points after the treatment with E64, T7 and T4.