The cells were infected with late-stage AG83 strain with variable membrane binding affinities

The cells were infected with late-stage AG83 strain with variable membrane binding affinities. evaluated their inhibitory efficacy against the AG83 strain of inhibitory activity of Glycoside-2 (Gly2) (1.13 M IC50 value) on promastigote demonstrated maximum cytotoxicity with ~94% promastigote death as compared to amphotericin B that was taken as a positive control. The antiproliferative effect of Gly2 on promastigote encouraged us to analyze the structureCactivity relationship of Gly2 with Gp63, a zinc metalloprotease that majorly localizes at the surface of the promastigote and has a role in its development and multiplication. The result demonstrated the exceptional binding affinity of Gly2 toward the catalytic domain of Gp63. These data were thereafter validated through cellular thermal shift assay in a Hexaminolevulinate HCl physiologically relevant cellular environment. Mechanistically, reduced multiplication of promastigotes on treatment with Gly2 induces the destabilization of redox homeostasis in promastigotes by enhancing reactive oxygen species (ROS), coupled with depolarization of the mitochondrial membrane. Additionally, Gly2 displayed strong lethal effects on infectivity and multiplication of amastigote inside the macrophage in the amastigoteCmacrophage infection model as compared to amphotericin B treatment. Gp63 is also known to bestow protection against complement-mediated lysis of parasites. Interestingly, Gly2 treatment enhances the complement-mediated lysis of promastigotes in serum physiological conditions. In addition, Gly2 was found to be equally effective against the clinical promastigote forms of PKDL strain (IC50 value of 1 1.97 M); hence, Hexaminolevulinate HCl it could target both VL and PKDL simultaneously. Taken together, this study reports the serendipitous discovery of Gly2 with potent antileishmanial activity and proves to be a novel chemotherapeutic prototype against VL and PKDL. spp. proteases involved in parasite life cycle and pathogenesis. Notably, leishmanolysin of spp., the known zinc metalloprotease, Gp63 has been identified as an important multifunctional virulence factor, found in abundance on the surface of promastigotes and in limited quantities inside the parasite (Etges et?al., 1985; Yao et?al., 2003; Sunter and Gull, 2017). It is the key enzyme responsible for parasite propagation, promastigote binding and internalization in macrophages, and attenuation of reactive oxygen intermediate formation that favors amastigote proliferation (Kamhawi, 2006). The myriad diversity as well as the high catalytic activity at the bodys physiological temperature of this virulence factor favors the dissemination of the parasite in the host (Chaudhuri and Chang, 1988; Yao et?al., 2002; McGwire et?al., 2003; Yao et?al., 2003). Recent reports from certain parasitic models demonstrated the protective nature of Gp63 against complement fixation and processing that shields promastigotes during its tarriance into mammalian hosts (Brittingham et?al., 1995; Joshi et?al., 1998; Joshi et?al., 2002). Emerging studies have shown that plant-derived glycoside formulations from Asteraceae extracts can inhibit protozoan parasites such as development, thus proposed as promising transmission-blocking sugar baits. Additional study has identified promising antibacterial and antiparasitic activity of oleanolic acid and its glycosides isolated from marigold (promastigote and intra-macrophagic amastigote forms. Gly2 treatment led to abrogation of parasite multiplication, induction of reactive oxygen species (ROS) generation, and disruption of mitochondrial Hexaminolevulinate HCl membrane potential leading to promastigote death, suggesting its therapeutic implication against VL. Hexaminolevulinate HCl Besides its inhibitory role in cultured parasites model mimicking the bodys physiological condition. The structureCactivity relationship Hexaminolevulinate HCl (SAR) analysis of novel glycosides, along with cellular thermal shift assay (CETSA) and studies, revealed efficient binding of Gly2 molecule with Gp63 catalytic domain. Additionally, Gly2 demonstrated excellent antileishmanial activity against the clinical isolate of PKDL. Overall, our work has discovered a purely non-toxic glycoside derivative with strong antileishmanial potential. Materials and Methods Parasite Growth and Maintenance Promastigote forms of (MHOM/IN/1983/AG83) were cultured at 26C in M199 (GIBCO, India), pH 7.4, supplemented with 10% (v/v) inactivated fetal bovine IL18R antibody serum (FBS, GIBCO, India) and 0.02 mg/ml gentamycin (Life Technologies, USA). The clinical isolate of PKDL, BS12, was obtained as a gift from Prof. Mitali Chatterjee [Department of Pharmacology, Institute of Postgraduate Medical Education and Research (IPGMER), Kolkata, India]. BS12 was routinely cultured at 22C in M199 (GIBCO, India) with 100 U/ml penicillin-streptomycin (Gibco, Invitrogen, Thermo Fisher Scientific, NY, USA), 8 M hemin (4 mM stock made in 50% triethanolamine) (Sigma, USA), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), supplemented with 10% heat-inactivated FBS (GIBCO, India). Cultures were maintained between 106 and 107 cells/ml for continuous.