Identification of Sulfated Polysaccharides with Activity against Plasmodium falciparum Cytoadherence Proteins using Targeted Sequencing and In Silico Characterisation

Abstract

Malaria is a global health problem that remains a leading cause of death especially in Sub-Saharan Africa. Plasmodium parasites in the human host undergo different stages with survival tactics to evade immune system clearance. One crafty mechanism of survival is cytoadherence, through which the parasite attached to human erythrocytes thus avoids splenic clearance. Cytoadherence is mediated by Merozoite surface and exported proteins. Diversity exhibited by these proteins is one of the main challenges in developing drugs targeting intracellular parasites. This study aimed at identifying sulfated polysaccharides with ability to block these proteins by investigating two drug targets as representatives: Merozoite surface protein with Duffy Binding-Like Domain (DBLMSP2) and Plasmodium-Helical Interspersed Subtelomeric domain b containing Ring-infected Erythrocyte Antigen (RESA) Like Protein (PHISTb/RLP1). Sanger sequencing was used to obtain sequences of these drug targets from whole blood samples collected from malaria-endemic zones in Kenya. The sequencing employed novel primers designed for the target genes from Primer3 plus. Reference sequences were obtained from Plasmodb database from 3d7 strain and used for genome mapping as well as structure comparisons. Functional Motifs were predicted using MEME software with default settings for consensus output. Protein structures were modelled using I-TASSER and LOMETs tools and validated using Galaxy Refine and RAMPAGE tools. Sulfated polysaccharides were screened from the Pub-Chem Compounds database. The criteria of selection were guided by the Lipinski rule of five for a drug compound. Interaction simulations were achieved using auto-dock vina. The docking results were visualized in PyMOL. Sequencing results were successful for the PHISTb/RLP1 gene but not for DBLMSP2. 157-point mutations were identified in PHISTb/RLP1 sequences and 12 of these single out for protein structure analysis. Protein structure prediction yielded high quality models passing >90% ramachandran score. PHISTb/RLP1 mutant structure folded differently compared to wild type structure. Eleven sulfated polysaccharides fulfilled the criteria of drug compounds. Alpha carrageenan, Amylopectin sulfate, cyclodextrine sulfate, ghatti sulfate, fucoidan and galactal interacted optimally with both drug targets after docking. Protein-Ligand interactions were different in wildtype PHISTb/RLP1 compared to wildtype inferring effects of mutations on drug binding. The study identifies lead compounds that can be added to malaria drug molecules pipeline. In vitro testing is recommended to obtain more data on inhibition capacity of the compounds. The study design has proven effective for initial stages of drug discovery and is recommended for other diseases as well.