singh, s. | bioRxiv Science

bioRxiv2020

Role of RBC membrane protein palmitoylation in regulation of molecular topology and susceptibility to Plasmodium falciparum invasion

Squeezability of biconcave RBC raises a fundamental query, about, how it can restructure its bendable cytoskeleton for efficient micro-circulation. We report for the first time, the existence of dynamic palmitoylome in RBC composed of 118 palmitoylated proteins that reduced to 42 upon treatment with 2BP, a generic inhibitor of palmitoylation. In-depth analysis revealed that Semaphorin7A, CR1 and ABCB6, the known RBC receptors for P. falciparum were reduced to negligible in 2BP-treated RBCs, suggesting palmitoylation-dependent recruitment of parasite-specific receptors. Interestingly, Kell, a single disulphide-linked co-partner in Kell-Kx complex was undetected in 2BP-treated RBCs, while Kx remained intact. RBCs-blocked with anti-Kell antibody demonstrated signficant reduction in parasite invasion, thus suggesting it as a receptor proto-type for P. falciparum invasion. Finally, reduced expression of Kell in palmitoylated protein pool of sickle-cell RBC ghost, with its diminished surface representation in these RBCs, proposed Kell, as one of the novel receptor-prototype for P. falciparum invasion.

cell biology↗

bioRxiv2020

Development of novel anti-malarial from structurally diverse library of molecules, targeting plant-like Calcium Dependent Protein Kinase 1, a multistage growth regulator of P. falciparum

Upon Plasmodium falciparum merozoites exposure to low [K+] environment in blood plasma, there is escalation of cytosolic [Ca2+] which activates Ca2+-Dependent Protein Kinase 1 (CDPK1), a signaling hub of intra-erythrocytic proliferative stages of parasite. Given its high abundance and multidimensional attributes in parasite life-cycle, this is a lucrative target for desiging antimalarials. Towards this, we have virtually screened MyriaScreenII diversity collection of 10,000 drug-like molecules, which resulted in 18 compounds complementing ATP-binding pocket of CDPK1. In vitro screening for toxicity in mammalian cells revealed that these compounds are non-toxic in nature. Further, SPR analysis demonstrated differential binding affinity of these compounds towards recombinantly purified CDPK1 protein. Selection of lead compound 1 was performed by evaluating their inhibitory effects on phosphorylation and ATP binding activities of CDPK1. Further, in vitro biophysical evaluations by ITC and FS revealed that binding of compound 1 is driven by formation of energetically favorable non-covalent interactions, with different binding constants in presence and absence of Ca2+, and TSA authenticated stability of compound 1 bound CDPK1 complex. Finally, compound 1 strongly inhibited intra-erythrocytic growth of P. falciparum in vitro. Concievably, we propose a novel CDPK1-selective inhibitor, step towards developing pan-CDPK kinase inhibitors, prerequisite for cross-stage anti-malarial protection.

biochemistry↗

bioRxiv2019

Supression of Perforin-like Protein pores inhibit Plasmodium multistage-growth, transmission and erythrocyte senescence

The pore forming Plasmodium perforin like proteins (PPLP), expressed in all stages of the parasite life cycle are central drivers for host interactions critical for completion of parasite life cycle and high transmission rates. The high sequence similarity in the central membrane attack complex/ perforin (MACPF) domain and consequent functional overlaps defines them as an attractive target for the development of multi-stage antimalarials. Herein we evaluated the mechanism of pan active function of central, highly conserved region of PPLPs, MACPF domain (PMD) and inhibitory potential of specifically designed anti-PMD chemo. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to haemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence at 48 hours which can account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing anemia during malaria infection. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress of merozoites and protecting against erythrocyte senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage and transmission-blocking potential of these inhibitors. Additionally, the erythrocyte senescence protective potential of PMD inhibitors can be used to occlude PPLPs mediated severe malarial anemia. Further these inhibitors can be developed with a potential to protect against severity of the disease.\n\nAuthor SummaryMalaria continues to be a major global health threat despite of several exciting improvements in the treatment and prevention of the disease. One of the major concerns in the development of therapy is the emergence of the drug resistance. But for the efficient treatment regime, targeting multiple stages including host and vector would serve as an ideal therapy. Perforin like proteins (PLPs) are eukaryotic pore forming proteins that are highly conserved in the apicomplexan parasites. These play crucial roles in entry and exit of parasites from the host cells and establish infection at multiple stages of Plasmodium spp. life cycle. Understanding the mechanism of pore formation by smaller, functional, pan-active scaffold of PLPs can serve as a target for development of cross stage protection. Here, using various biochemical, biophysical and pharmacological evidences, we validate the activity and characterize the pore formation of PLPs on erythrocytes. Further, our specifically designed inhibitors could restrict this pore formation and impede the exit/entry of the parasites. Moreover, these inhibitors could also exert multiple stage inhibition and rescue the uninfected erythrocytes from death. Together, this study highlights the mechanism of pore formation by PPLPs and evaluates their potential for the development of pan-active inhibitors to provide both symptomatic and transmission blocking cure for malaria.

molecular biology↗