Biochemical characterization and chemical validation of Leishmania MAP Kinase-3 as a potential drug target

Abstract

Protozoan parasites of the Leishmania genus have evolved unique signaling pathways that can sense various environmental changes and trigger stage differentiation for survival and host infectivity. MAP kinase (MAPK) plays a critical role in various cellular activities like cell differentiation, proliferation, stress regulation, and apoptosis. The Leishmania donovani MAPK3 (LdMAPK3) is involved in the regulation of flagella length and hence plays an important role in disease transmission. Here, we reported the gene cloning, protein expression, biochemical characterizations, inhibition studies and cell proliferation assay of LdMAPK3. The recombinant purified LdMAPK3 enzyme obeys the Michaelis-Menten equation with Km and Vmax of LdMAPK3 was found to be 20.23 nM and 38.77 ± 0.71 nmoles ATP consumed/mg LdMAPK3/min respectively. The maximum kinase activity of LdMAPK3 was recorded at 35 °C and pH 7. The in-vitro inhibition studies with two natural inhibitors genistein (GEN) and chrysin (CHY) was evaluated against LdMAPK3. The Ki value for GEN and CHY were found to be 3.76 ± 0.28 µM and Ki = 8.75 ± 0.11 µM respectively. The IC50 value for the compounds, GEN and CHY against L. donovani promastigotes were calculated as 9.9 µg/mL and 13 µg/mL respectively. Our study, therefore, reports LdMAPK3 as a new target for therapeutic approach against leishmaniasis.

Leishmania Protein Kinases: Important Regulators of the Parasite Life Cycle and Molecular Targets for Treating Leishmaniasis

“…On the other hand, Lmx MPK3 deletion mutants had a shorter flagellum ( Figure 2 ) [ 123 ], a similar phenotype to null mutants of Lmx MPKK1, the activating kinase of Lmx MPK3 [ 125 ]. Although Lmx MPK3 is not essential for parasite viability, small molecule inhibitors have been identified, as this kinase is important for Leishmania transmission [ 80 ]. Shweta Raj et al showed that the plant-based natural compounds genistein and chrysin ( Table 1 ) inhibited the kinase, and at the same time, minimized the parasitic load of infected macrophages ( Figure 2 ) [ 80 ].…”

The ultimate fate determinants of drug induced cell-death mechanisms in Trypanosomatids

“…Although MAPK-dependent pathways are one of the major cellular signaling cascades in mammals, it has scarcely been studied in Trypanosomatids. In a recent study, genistein (GEN) and chrysin (CHY) in a dose-dependent manner could inhibit the kinase activity of LdMAPK3 and thus exhibit anti-leishmanial activity ( Raj et al., 2019 ). In another study, LdMAPK1 was down-regulated in antimony-resistant clinical isolates, and susceptibility to antimony increased upon overexpression of LdMAPK1 in these parasites.…”

Multiple targets of balancing selection inLeishmania donovanicomplex parasites

“…LdBPK_363870.1 ( Figure 3 ) encodes a protein with similarity to mitogen-activated protein (MAP) kinases, a large family of enzymes implicated in antimony drug resistance (Ashutosh et al , 2012;Raj et al , 2019) . While candidacy was indicated by BS tests only in the East African population EA1, nucleotide diversity is also high in ISC1,2 and EA2 populations ( Supplementary Table 2 ).…”

“…LdBPK_363870.1 ( Figure 3 ) encodes a protein with similarity to mitogen-activated protein (MAP) kinases, a large family of enzymes implicated in antimony drug resistance (Ashutosh et al , 2012;Raj et al , 2019 and therefore an endomembrane member of the STE family (Baker et al , 2020) .…”