Unveiling Prospective Therapeutic Potential of Conserved Hypothetical Plasmodium falciparum Proteins by Using Integrated Proteo Genomic Annotation and In-Silico Therapeutic Discovery Approach.

Abstract

The increasing incidence of malaria and the emergence of drug-resistant strains highlight the critical need for new therapeutic targets. A recent study employing saturation mutagenesis has identified several essential, conserved genes in Plasmodium falciparum that code for proteins with unknown functions, presenting potential new avenues for therapeutic intervention. We hypothesized that these essential conserved hypothetical proteins could be functionally annotated with therapeutic relevance using an in-silico framework. However, a comprehensive framework for the functional annotation and classification of potential drug and vaccine candidates using in-silico tools has not been well established. While approaches like proteomics, subtractive genomics, and transcriptomics offer valuable insights, their isolated application limits the thorough functional annotation of proteins, and many studies do not explore therapeutic potential fully. To address these gaps, we developed the Integrated ProteoGenomic Annotation Framework (IPGAF), an in-silico protocol designed to annotate hypothetical proteins and screen them for druggability and antigenicity. Our IPGAF framework employs a two-step methodology. The first step focuses on functional annotation, integrating Pfam score-based domain analysis, orthology inference for evolutionary insights, functional linkage evaluation, subcellular localization prediction, domain architecture identification, and protein-protein interaction analysis. The second step assesses the potential of these proteins as drug targets or vaccine candidates through physicochemical and virulence evaluation, antigenicity prediction, identification of non-homologous proteins relative to the human proteome, druggability prediction, molecular docking studies, and the identification of multiple immunogenic regions (B cell, T cell, HLA) for multiepitope vaccine design. Using the IPGAF framework, we annotated 14 conserved hypothetical P. falciparum proteins from an initial set of 44. Among them, PF3D7_1208100, a merozoite protein, emerged as a promising drug and vaccine target, while PF3D7_0703900 and PF3D7_0916400 showed strong druggability potential. Our vaccine study identified the VC6 construct, incorporating epitopes from PF3D7_1223500, PF3D7_1348400, PF3D7_1470100, and PF3D7_1208100, as the most promising candidate due to its high antigenicity, non-allergenicity, and favourable physicochemical properties. Further in vitro validation could confirm the therapeutic potential of these proteins.