Differential affinity chromatography coupled to mass spectrometry: a 3R-relevant tool to identify common binding proteins of a broad-range antimicrobial peptide and potentially other compounds active against Toxoplasma gondii

Abstract text

Introduction: Leucinostatins and derivatives are antimicrobial peptides (AMPs) with broad-range of activities against cancer cells and infectious agents. One of these, ZHAWOC_6027 (AMP6027), was previously reported as a promising treatment option for African Sleeping Sickness (Brand et al. Angew Chem Int Ed Engl 2021, 60)

Objective: To investigate the efficacy of AMP6027 against Toxoplasma gondii in vitro and in vivo, and to identify potential drug targets.

Materials & methods: In vitro efficacy (EC50) and ultrastructural studies were done using T. gondiitachyzoites grown in human foreskin fibroblasts (HFF). For in vivo studies, AMP6027 (3 mg/kg/day for 5 days) was applied s. c. in mice experimentally infected with T. gondii oocysts, and cerebral parasite load was determined by real time PCR. The effects of peptide 6027 on B and T cells were investigated in murine splenocytes. To identify peptide 6027-binding proteins in Toxoplasma and splenocytes, differential affinity chromatography coupled to mass spectrometry and proteomics (DAC-MS-proteomics) was performed with cell-free extracts of T. gondii tachyzoites and mouse spleens using AMP6027 or an ineffective analogue.

Results: AMP6027 was highly efficacious in vitro (EC50 = 2 nM), and induced distinct mitochondrial changes as seen by TEM. HFF EC50 was 1000 x higher. In vivo, however, AMP6027 exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. AMP6027 also impaired the proliferation and viability of LPS-stimulated B cells in vitro. Following DAC, MS and proteomics of T. gondii extracts, 269 proteins were identified binding specifically to AMP6027, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved mitochondrial energy metabolism and in protein processing and secretion, and in T. gondii, the cytochrome oxidase (TgApiCox25) complex was identified as the main molecular target among others These results suggest that AMP6027 interacts with common targets involved in essential pathways.

Conclusions: Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC-MS-proteomics should be considered a smart and 3R-relevant way to (i) define putative drug targets in pathogens, and (ii) eliminate compounds that are prone to potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans.