Computational study, synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colorectal cancer cells

Abstract

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 KDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and β-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer –therapeutic agents.

Site-Directed Mutants of Parasporin PS2Aa1 with Enhanced Cytotoxic Activity in Colorectal Cancer Cell Lines

“…These modifications allowed us to build a library of new parasporins with different activities against colorectal cancer cell lines. Site-directed mutagenesis was performed considering the results of Cruz et al [ 6 ], where peptides from loop 1 of PS2Aa1 had remarkable activity and adherence with SW480. It was established that the oligonucleotides with the mutations incorporated for site-directed mutagenesis should be present at the N-terminal end (variable region), specifically in domain I of PS2Aa1 because, based on previous molecular docking studies, this region is the one presumably responsible for specific binding to membrane receptors [ 14 ].…”

“…Mizuki et al, in the first report on Parasporin (PS) in 2000 [ 5 ], described its potential as an anticancer molecule, specifically in leukemia. Subsequently, most studies have focused on the characterization and screening of new proteins identified from Bacillus thuringiensis ( Bt ) strains and isolates [ 6 , 7 , 8 , 9 , 10 ]. Akiba and Okumura proposed in 2017 that the mechanism of action of this protein is in inducing apoptosis [ 11 ], but many unknowns persist up to now, especially regarding the identity of the major PS receptor, with N-aminopeptidase (APN) and GPI-anchored proteins having been proposed as candidates [ 6 , 7 , 12 , 13 ].…”

“…Subsequently, most studies have focused on the characterization and screening of new proteins identified from Bacillus thuringiensis ( Bt ) strains and isolates [ 6 , 7 , 8 , 9 , 10 ]. Akiba and Okumura proposed in 2017 that the mechanism of action of this protein is in inducing apoptosis [ 11 ], but many unknowns persist up to now, especially regarding the identity of the major PS receptor, with N-aminopeptidase (APN) and GPI-anchored proteins having been proposed as candidates [ 6 , 7 , 12 , 13 ]. Parasporin 2Aa1 (PS2Aa1) has shown cytotoxic effects against several human cancer cells, being highly specific for human liver and colon cancer cells [ 7 , 8 , 9 ], thus representing an additional treatment alternative for colorectal cancer.…”

“…In that study, the Loop1–PS2Aa and P264–G274, peptides exhibited stronger anticancer activity than the wild type against SW480 and SW620, respectively, and demonstrated high effectiveness and selectivity; hence, they were proposed as possible alternative therapeutic agents for the treatment of colon cancer. In this sense, we decided to take domain I of PS2Aa1, mutate the residues that presented differentiated activities in the reported peptides, and consider additional positions for the modification of these peptides to generate the libraries of PS2Aa1 mutants, which we describe below [ 6 ]. The goal of this work was to obtain a characterized set of PS2Aa1 mutants with modifications in domain I and assess its cytotoxicity in colon cancer cells.…”

Chemoinformatics and Artificial Intelligence Colloquium: Progress and Challenges to Develop Bioactive Compounds

“…Other chemoinformatics, ML, and DL models were proposed to counteract the antibiotic resistance found in all parts of the world [68], and peptides have been proposed as alternatives. In general, peptides have biological activity against bacteria, viruses, fungi, and parasites [69,70].…”