Computational and experimental elucidation of Plasmodium falciparum phosphoethanolamine methyltransferase inhibitors: Pivotal drug target

Abstract

Introduction Plasmodium falciparum synthesizes phosphatidylcholine for the membrane development through serine decarboxylase–phosphoethanolamine methyltransferase pathway for growth in human host. Phosphoethanolamine-methyltransferase ( Pf PMT) is a crucial enzyme for the synthesis of phosphocholine which is a precursor for phosphatidylcholine synthesis and is considered as a pivotal drug target as it is absent in the host. The inhibition of Pf PMT may kill malaria parasite and hence is being considered as potential target for rational antimalarial drug designing. Methods In this study, we have used computer aided drug designing (CADD) approaches to establish potential Pf PMT inhibitors from Asinex compound library virtually screened for ADMET and the docking affinity. The selected compounds were tested for in-vitro schizonticidal, gametocidal and cytotoxicity activity. Nontoxic compounds were further studied for Pf PMT enzyme specificity and antimalarial efficacy for P . berghei in albino mice model. Results Our results have identified two nontoxic Pf PMT competitive inhibitors ASN.1 and ASN.3 with better schizonticidal and gametocidal activity which were found to inhibit Pf PMT at IC 50 1.49μM and 2.31μM respectively. The promising reduction in parasitaemia was found both in orally (50 & 10 mg/kg) and intravenous (IV) (5& 1 mg/kg) however, the better growth inhibition was found in intravenous groups. Conclusion We report that the compounds containing Pyridinyl-Pyrimidine and Phenyl-Furan scaffolds as the potential inhibitors of Pf PMT and thus may act as promising antimalarial inhibitor candidates which can be further optimized and used as leads for template based antimalarial drug development.

Progress and challenges in the use of fluorescence‐based flow cytometric assays for anti‐malarial drug susceptibility tests

“…There are several methods for the assessment of intraerythrocytic Plasmodium growth: morphological observation under a microscope and biochemical measurements. For morphology-based assays, the microtechnique developed by Rieckmann et al [3] is simple and reliable and has still been used in recent reports [13][14][15][16][17][18]. For biochemical assays, the microtechnique was modified to a semiautomatic tritiated hypoxanthine incorporation assay, measuring Plasmodium uptake of radiolabelled hypoxanthine, a nucleic acid precursor, to assess the growth inhibitory effect of anti-malarial drugs [19] or immune serum [20].…”

A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

“…S2 B). To determine whether TTC0109 had arsenite methyltransferase activity, a coupled spectrophotometric enzymatic assay based on the formation of S-adenosylhomocysteine (SAH) from SAM after transfer of methyl-group(s) on the substrate was employed (33,34). In this case, the acceptor of methyl groups was As(III).…”

“…According to the manufacturer's protocol, the TtArsM arsenite methyltransferase activity was measured using the SAM510: SAM Methyltransferase Assay Kit (G-Biosciences) with modifications regarding the temperature, the SAM concentration and the reaction time (33)(34)(35). The assay relies on the degradation…”

“…copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights The this version posted July 14, 2021. ; https://doi.org/10.1101/2021.07.12.452139 doi: bioRxiv preprint activity was measured using the SAM510: SAM Methyltransferase Assay Kit (G-Biosciences) with modifications regarding the temperature, the SAM concentration and the reaction time (33)(34)(35). The assay relies on the degradation of S-adenosylhomocysteine (SAH) into urate and hydrogen peroxide by a mixture of enzymes (adenosylhomocysteine nucleosidase, adenine deaminase, xanthine oxidase).…”

“…with kanamycin (50 µg/mL) and chloramphenicol(33 µg/mL): i) E. coli BL21-CodonPlus (DE3)-RIL: pET30/TtarsM, ii) E. coli BL21-CodonPlus (DE3)-RIL: pET30/ TtarsM C77S, iii) E. coli BL21-CodonPlus (DE3)-RIL: pET30/ TtarsM H40A, iv) E. coli BL21-CodonPlus (DE3)-RIL: pET30/ TtarsM H179A and v) E. coli BL21-CodonPlus (DE3)-RIL: pET30 (control). The pre-cultures were incubated at 37°C for 16 h at 180 rpm.…”