Multistage antiplasmodial activity of hydroxyethylamine compounds, in vitro and in vivo evaluations

Abstract

Malaria, a global threat to the human population, remains a challenge partly due to the fast-growing drug-resistant strains of Plasmodium species. New therapeutics acting against the pathogenic asexual and sexual stages, including liver-stage malarial infection, have now attained more attention in achieving malaria eradication efforts. In this paper, two previously identified potent antiplasmodial hydroxyethylamine (HEA) compounds were investigated for their activity against the malaria parasite's multiple life stages. The compounds exhibited notable activity against the artemisinin-resistant strain of P. falciparum blood-stage culture with 50% inhibitory concentrations (IC₅₀) in the low micromolar range. The compounds' cytotoxicity on HEK293, HepG2 and Huh-7 cells exhibited selective killing activity with IC₅₀ values > 170 μM. The in vivo efficacy was studied in mice infected with P. berghei NK65, which showed a significant reduction in the blood parasite load. Notably, the compounds were active against liver-stage infection, mainly compound 1 with an IC₅₀ value of 1.89 μM. Mice infected with P. berghei sporozoites treated with compound 1 at 50 mg kg^-1 dose had markedly reduced liver stage infection. Moreover, both compounds prevented ookinete maturation and affected the developmental progression of gametocytes. Further, systematic in silico studies suggested both the compounds have a high affinity towards plasmepsin II with favorable pharmacological properties. Overall, the findings demonstrated that HEA and piperidine possessing compounds have immense potential in treating malarial infection by acting as multistage inhibitors.

Fig. 1. (A) Structure of compounds 1 and 2; (B) cytotoxicity data for Alamar blue assay (IC₅₀) were transformed, X = log₍₁₀₎, normalized between 0% and 100% and fitted with sigmoidal dose–response curves; (C) anti-plasmodial activity of compounds against ART-resistant strain, data (relative SYBR green-1 fluorescence) were transformed, X = log₍₁₀₎, normalized between 0% and 100% and fitted with sigmoidal dose–response curves. DHA – dihydroartemisinin, CQ – chloroquine.

Fig. 2. Assessment of the efficacy of compounds (1 and 2) against P. berghei NK-65 infection in the mouse model; (A) the mean percentage parasitemia of treated group versus untreated ones on days 5, 8, and 15 post-treatment. The data represent means ± SD using five animals (n = 5) in each group; (B) the survival response of mice during blood-stage infection and mean survival time (MST) (in days) treated versus untreated ones; (C) efficacy of compound 1 at 50 mg kg⁻¹ (i.p) against P. berghei liver stage infection was compared with control mice using unpaired t-test (P < 0.02); and (D). The survival analysis of mice in liver-stage infection was monitored for compound 2 until day 30 post-infection (n = 4 per group).

Fig. 3. (A) Each scatters plot column represents different sets of samples from different time points and treatments. A 100 μL 3^rd, 4^th and 5^th-day gametocyte rich culture was seeded in microtiter plates in triplicates for each treatment (0.5 μM) and control and three wells each was fixed and stained by the Giemsa staining. Both compounds had an apparent reduction of intact gametocytes compared to control; (B) the most representative morphologies were documented. Both compounds had an evident detrimental effect on the normal morphology to the extent that none of the treated samples had any mature gametocytes even though with 50% viability in SYBR green screenings.

Fig. 4. Structural changes in the sexual stage of malaria. Efficacy of compound 1 (A) and 2 (B) against ookinete formation; (C) inhibition curve for both the compounds; (D) representative images of the DMSO control (healthy) ookinete and treatment with both the compounds at different concentrations. The photomicrograph of smears was stained with Giemsa shown at 1000× magnification.

Fig. 5. (A and B) shows pharmacophoric (red, not seen), antipharmacophore (blue), and ballast part (grey) in compounds; however, (C and D) show toxicophoric (red) and non-toxicophoric (blue) part in compounds.

Fig. 6. Docked images. (A) Compound 1 with PLM 2; (B) compound 2 with PLM 2.

Fig. 7. MD simulations based generated plots showing the calculated distances between the PLM and the designed inhibitors (A; top, left) and (B; top, right). Highlighted the changes in hydrogen bonds (C and D). Red and violet color represent compound 1 and 2, respectively.

Fig. 8. The observed structural changes with (A, top) were showing the variation in the system compactness in terms of R_g values (B, lower), illustrating the changes in the RMSD values observed during 100 ns MD simulations.

Fig. 9. The MM/PBSA based dynamic pattern of calculated interaction energies highlighted in the form of the 2-D curves with (A) observed variation in the compound 1 and PLM II complex: (B) quantified values for compound 2 and PLM II system.