An Integrated Approach of Proteomic, Cheminformatic, and In Vitro Drug Screening to Identify Potential Therapeutic Targets of Taenia solium Cysticerci Proteases

Abstract

Proteases are primary enzymes that are involved in parasites' host invasion by disrupting the physical barriers and immune system. Due to the mass anthelminthic drive against helminths by WHO, the occurrence and spread of drug resistance strains have increased. Taenia solium is one of the major helminthic infections to humans. The CNS infection of parasites' cysts leads to epilepsy, i.e., neurocysticercosis. The existing drugs are several decades old, and their efficacy is poor for the treatment of CNS infection. This demands one to look for new drugs for use in the near future. Detailed proteomic characterization of T. solium proteases is lacking, though they could be one of the primary targets for the development of new drugs. We used LC-MS/MS-based proteomics to characterize and identify proteases, bioinformatics to identify essential proteases, and cheminformatics tools to screen their inhibitors from the ZINC15 drug bank. Finally, we did in vitro cytotoxicity, inflammatory, and cysticidal assays for drug repurposing. We identified proteases present on cyst fluid (n = 50) and cyst wall (n = 23) and sorted seven essentials, ones that were used for docking studies with drugs. The docking studies led to five preapproved drugs, and finally two were sorted. We report 100% efficacy with 1,10-phenanthroline (metalloprotease inhibitor) and belinostat in in vitro cysticidal experiments. 1,10-Phenanthroline had better blood-brain permeability and was less cytotoxic when compared with albendazole sulfoxide and praziquantel. Thus, we propose 1,10-phenanthroline as an alternative drug for NCC. However, this needs further detailed investigation in a suitable animal model.

1

GO enrichment analysis of proteases present in the Taenia solium cyst wall (CW) and cyst fluid (CF). (A) Top enriched molecular functions. (B) Top enriched biological functions. (C) Top enriched cellular components. Ordinates on the Y-axis represent the GO categories.

2

Phylogenetic analysis of the proteases present in Taenia solium (A) cyst fluid (CF) and (B) cyst wall (CW) showing a high sequence similarity between them.

3

Docking and hydrogen bond formation between essential proteases of T. solium and FDA-approved drugs using AutoDock Vina and LigPlot software, respectively. (A) Leukotriene A4 hydrolase, (B) dipeptidyl-peptidase III, (C) carboxypeptidase E, (D) acidic peptidase 2, (E) deglycase, (F) cathepsin L, and (G) cyclotransferase.

4

Interaction studies of phenyl vinyl sulfone and 1,10-phenanthroline protease inhibitors with identified and essential proteases of T. solium. (A) Leukotriene A4 hydrolase, (B) dipeptidyl-peptidase III, (C) carboxypeptidase E, (D) cathepsin L, and (E) deglycase (red dotted lines represents hydrophobic interactions, and green dotted lines represent hydrogen bonds).

5

Inhibition of parasitic proteases by inhibitors and drugs to identify their possible roles in their survival. Viable cysts were treated with (A) drugs at their IC50 value and (B) protease inhibitors with 50 μg/mL for 24 h and then stained with trypan blue dye to know the viability of the treated cysts. Phenyl vinyl sulfone and 1,10-phenanthroline were most effective among the inhibitors and drugs with 100% killing efficacy. Images are representative for three independent biological experiments.

6

Cytotoxic effect of phenyl vinyl sulfone (PVS) and 1,10-phenanthroline on HEK293T (A, C) and HepG2 (B, D) cell lines compared with anthelminthic drugs (albendazole sulfoxide and praziquantel) at various concentrations (A, B) and dilutions of IC50 values (C, D). 1,10-Phenanthroline showed least cytotoxicity in every condition. Dose-dependent study of 1,10-phenanthroline to identify the minimum inhibitory concentration of protease inhibitor (E). Images are representative for three independent biological experiments.

7

Relative gene expression analysis of pro- and anti-inflammatory cytokines in three different cell lines of 1,10-phenanthroline (25 μg/mL) by QPCR. (A) dTHP-1 cells, (B) HepG2 cells, and (C) HEK293T cells. 1,10-Phenanthroline showed promising potency of suppressing pro-inflammatory cytokines in comparison to albendazole and praziquantel. Images are representative for three independently performed biological triplicates. Statistical analysis as *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005 are compared as indicated in the figures. Images are representative for three independent biological experiments.