Bazhenov, S. | Search bioRxiv

bioRxiv2025

Correlative evaluation of anticancer effects of a modified methioninase MGL-KPV using 2D and 3D cell models, human cancer xenografts in zebrafish embryos and Balb/c nude mice

PurposeThe emergence of new models of tumor growth requires a comparison of the results of the evaluation of the anticancer effect of single drug candidate for the selection of a relevant model. MethodsWe assessed the anticancer activity of the modified enzyme MGL-KPV using four models: 2D cultures, 3D spheroids, zebrafish embryo xenografts, and Balb/c nude mice. Cytotoxicity was tested in cancer cell lines and fibroblasts; efficacy was evaluated in zebrafish and mice. The effect of MGL does not require direct contact with cancer cells, making it unique drug candidate for in vitro and in vivo comparison. ResultsMGL-KPV showed selective cytotoxicity with IC50 values of 0.23-2.21 U/mL (2D) and 1.43-4.82 U/mL (3D), sparing fibroblasts. HCT-116 and Panc-1 were most sensitive. In zebrafish xenografts, tumor size reduced by 70% (HCT-116) and 43% (Panc-1); in mice -- by 45% and 34%, respectively. ConclusionsMGL-KPV demonstrated significant anticancer activity across all models. These results support step-by-step non-clinical proof-of-concept studies approach for amino-acid cleaving enzymes with anticancer properties.

pharmacology and toxicology↗

bioRxiv2025

Structural and Functional Characterization of Encapsulin-Targeted Double Ferritin Fold Ferroxidases

Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four -helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/676036v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@72e6b0org.highwire.dtl.DTLVardef@18752fforg.highwire.dtl.DTLVardef@15a02e7org.highwire.dtl.DTLVardef@74cd02_HPS_FORMAT_FIGEXP M_FIG C_FIG A family of double ferritin-like proteins (DFLPs) composed of two four-helical domains is described and investigated. DFLPs are shown to form homodimers, bind heme, possess diiron sites and display ferroxidase activity. Some DFLPs are shown to be targeted to encapsulin shells as core or secondary cargo, thus representing a new type of iron metabolism systems.

biochemistry↗