Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein

Abstract

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds. Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modeled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: This research identifies inhibitors of exported proteins that can be used in in vitro tests against the Plasmodium parasite.

Keywords: Anti-malarials; Exported proteins; Interactions; P. falciparum; PHISTb/RLP1; Sulfated polysaccharides.

Figure 1.. High frequency non-synonymous SNPs observed in sequenced data for PHISTb/RLP1 protein.

Non-synonymous SNPs occurring in 50% and above of the total sample size. Codons I129T, T142V, Y147D, E154Q, S156H, T167I, S208L, M211T, L219H, D387N, D390N, and E403K were substitution of amino acids across different functional groups. These were modelled and their implications on protein structure and interactions with a drug compound investigated.

Figure 2.. PHISTb/RLP1 reference and mutant homology modelled structures and their predicted binding sites clusters.

( a) PHISTb/RLP1 reference protein structure shown in green; ( b) structure of PHISTb/RLP1 mutant protein structure shown in cyan; ( c) PHISTb/RLP1 reference protein binding site clusters, site 1 in red, site 2 in blue and site 3 in yellow; ( d) PHISTb/RLP1 mutant protein binding sites clusters, site 1 in pink, site 2 in orange and site 3 in dark purple. All models visualized in PyMOL.

Figure 3.. A LigPlot showing interactions of the low molecular weight heparin compound with residues in the active site of the PHISTb/RLP1 reference protein.

The compound forms hydrophobic interactions with S161 and K205 amino acids. Other interactions shown include hydrogen bonding N197 S201 and E204 all within the PHISTb/RLP1 binding pocket.

Figure 4.. Visualization of protein-ligand interactions for both PHISTb/RLP1 reference and mutant proteins.

( a and b) visualizations of alpha carrageenan (firebrick) and fucoidan (blue) drug compounds interacting with PHISTb/RLP1 reference protein structure (green) at specific binding sites. The interacting residues and polar contacts are shown in firebrick and blue PyMOL colours respectively. ( c and d) are visualizations of alpha carrageenan (firebrick) and ghatti sulfate (purple) compounds interacting with PHISTb/RLP1 mutant protein structure binding pockets. The interacting residues and polar contacts are shown in firebrick and purple PyMOL colours, respectively. These specific interactions reveal potential inhibitory activity of these compounds against PHISTb/RLP1 proteins.

Figure 5.. Ligplot showing hydrogen bonding, van der Waals forces and hydrophobic interactions between carrageenan and PHISTb/RLP1 reference protein.

The compound interacts with S132, R315 and N80 amino acids located in the protein model binding site. This reveals the high affinity of interaction of this compound to the protein target.