In silico directed evolution of Anabas testudineus AtMP1 antimicrobial peptide to improve in vitro anticancer activity

Abstract

Various studies have demonstrated that directed evolution is a powerful tool in enhancing protein properties. In this study, directed evolution was used to enhance the efficacy of synthesised Anabas testudineus AtMP1 antimicrobial peptides (AMPs) in inhibiting the proliferation of cancer cells. The modification of antimicrobial peptides (AMPs) and prediction of peptide properties using bioinformatic tools were carried out using four databases, including ADP3, CAMP-R3, AMPfun, and ANTICP. One modified antimicrobial peptide (AMP), ATMP6 (THPPTTTTTTTTTTTTTAAPARTT), was chosen based on its projected potent anticancer effect, taking into account factors such as amino acid length, net charge, anticancer activity score, and hydrophobicity. The selected AMPs were subjected to study in deep-learning databases, namely ToxIBTL and ToxinPred2, to predict their toxicity. Furthermore, the allergic properties of these antimicrobial peptides (AMPs) were verified by utilising AllerTOP and AllergenFP. Based on the results obtained from the database study, it was projected that antimicrobial peptides (AMPs) demonstrate a lack of toxicity towards human cells that is indicative of the broader population. After 48 hours of incubation, the IC₅₀ values of ATMP6 against the HS27 and MDA-MB-231 cell lines were found to be 48.03 ± 0.013 µg/ml and 7.52 ± 0.027 µg/ml, respectively. The IC₅₀ values of the original peptide ATMP1 against the MDA-MB-231 and HS27 cell lines were determined to be 59.6 ± 0.14 µg/ml and 8.25 ± 0.14 µg/ml, respectively, when compared. Furthermore, the results indicated that the injection of ATMP6 induced apoptosis in the MDA-MB-231 cell lines. The present investigation has revealed new opportunities for advancing novel targeted peptide therapeutics to tackle cancer.

Keywords: Anabas testudineus; Antimicrobial peptide; Breast cancer; Directed evolution; Peptide drug.

Figure 1. Workflow for bioinformatics prediction of antimicrobial peptides.

Figure 2. Cytotoxicity effect of synthetic ATMP5 on cancer MDA-MB-231 breast cancer cell line.

Figure 3. Apoptosis detection results by Annexin V FTIC-A assay.

This assay identifies apoptotic cells based on Annexin V binding, distinguishing between early apoptosis (Annexin V positive, PI negative) and late apoptosis (both Annexin V and PI positive) using the Elabscience E-CK-A211 kit. The results were also acquired with BD-FACSCanto II analyzers.

Figure 4. The cell cycle gene expression profile in MDA-MB-231 cells.