Joint research project

Engineered methionine gamma-lyase as a novel anticancer drug

Project leaders
Andrea Mozzarelli, Tatyana Demidkina
Agreement
RUSSIA - RFBR-suspended - Russian Foundation for Basic Research
Call
CNR/RFBR 2015-2017
Department
Physical sciences and technologies of matter
Thematic area
Physical sciences and technologies of matter
Status of the project
New

Research proposal

Methionine is an essential amino acid playing different and crucial roles in mammalian metabolism, such as protein synthesis, methylation of DNA carried out via S-adenosyl methionine, and polyamine synthesis. A methionine dependence in cancer cells has been observed and attributed to a combination of molecular events, including deletions, polymorphisms or alterations in the expression of genes in the methionine de novo and salvage pathways. Cancer cells with these defects are unable to regenerate methionine via these pathways and therefore depend for their growth on the methionine pool in the surrounding medium. Selective killing of methionine dependent cancer cells in co-culture with normal cells has been demonstrated using culture media deficient in methionine [Hoffman and Erbe (1976). PNAS 73, 1523-1527; Cavuoto and Fenech (2012) Cancer Treatment Reviews 38, 726-736].
The project exploits the "methionine dependence" of cancer cells for the development of a novel powerful anti-cancer therapy. The novel drug is based on methionine gamma-lyase (MGL), a bacterial enzyme that degrades methionine. Evidence of the therapeutic effect of MGL on cancer cells has been already reported [Tan et al. (2010) Anticancer Res. 30, 1041-1046; Amadasi et al. (2007) Curr. Med. Chem. 14, 1291-1324]. The milestones of the project are: i) the optimization of MGL for methionine cleavage by random and site-directed mutagenesis, ii) the pegylation of optimized MGL mutants and/or encapsulation in biodegradable matrices to increase bioavailability and to reduce immunogenicity, iii) the selective delivering to cancer cells by decorating the pegylated MGL or MGL nanocapsules with peptides and antibodies targeting cancer cells, and iv) the citotoxicity evaluation of pegylated MGL and MGL nanocapsules on cancer cell lines and in vivo.
MGL (EC 4.4.1.11) is a homotetrameric pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reaction of gamma-elimination of L-methionine to give methanethiol, alpha-ketobutyrate and ammonia. The catalytic mechanism and the the three dimensional structure have been determined and the catalytic activity has been assessed in the crystalline state [Ronda et al (2011) Biochim Biophys Acta. 1814 834-842]. MGL is absent in mammalian cells that exploit other enzymes for methionine degradation. Methionine depletion in the medium surrounding cancer cells leads to a decrease of cancer cells growth and to an increase in the susceptibility of cancer cells to drugs [Cavuoto and Fenech (2012) Cancer Treatment Reviews 38, 726-736].
The project aims at:
i) the increase of the catalytic activity of MGL by random and site-directed mutagenesis. The latter approach will be directed by the three dimensional structure of MGL determined by the Russian group (Revtovich, S.V., Faleev, N.G., Morozova, E.A., Anufrieva, N.V., Nikulin, A.D. & Demidkina, T.V. (2014). Biochimie. 101, 161-167). MGL mutants will be expressed, purified and characterized for catalytic efficiency and stability exploiting spectroscopic methods [Strambini et al. (1992) Biochemistry 31 7527-7534; Strambini et al. (1992) Biochemistry 31, 7535-7542; Cioni and Strambini (2002) Biophysical J. 82, 3246-3253; Cioni and Strambini (2002) Biochim. Biophys. Acta 1596,116-130; Gabellieri et al. (2011) Eur. Biophys. J. with Biophys, Letters. 40, 1237-1245]. Optimized mutants will be crystallized and their structures will be determined by X-ray crystallography;
ii) the improvment of MGL bioavalability, the prevention of MGL recognition by the immuno-system and proteases via conjugation with polyethylene glycol (PEG) and/or entrapment in biodegradable matrices. Optimized MGL mutants will be reacted with PEG of different molecular weight at lysine or cysteine residues [Pioselli et al. (2004) Protein Sci. 13 913-924; Caccia et al. (2009) Bioconjug. Chem. 20 1356-1366]. MGL-contained nanocapsules of polyacrylamide, polylactic acid, polyaminoacids and polysaccharides, eventually conjugated with PEG, will be prepared [Bruno et al. (2011) American Scientific Publishers, Ed. H. S. Nalwa. Encyclopedia of Nanoscience and Nanotechnology 21, 481-517]. The molecular weight of PEG molecules to be attached and the residues to be modified will be selected on the basis of a screening of different PEG and functionalized PEG MGL derivatives determining their catalytic activity and stability. Similarly, the most suitable matrix for MGL encapsulation will be selected on the basis of a screening of matrices determining enzyme catalytic activity and stability.
iii) the decoration of pegylated MGL mutants and MGL nanocapsules with peptides (Kappoor et al., PLoSONE, 2012, 7, e35187) or antibodies that specifically recognize cancer surface epitope, allowing specific targeting of cancer cells. Both peptides and antibodies can be acquired from commercial sources, and attached to pegylated MGL or MGL nanocapsules exploiting functionalized PEG and matrices;
iv) evaluation of the cytotoxic activity of pegylated MGL and MGL nanocapsules in the absence and presence of targeting peptides and antibodies on cancer cells cell lines of human tumors;
v) the labeling of pegylated MGL and MGL nanocapsules with fluorescent dyes for the detection of the interaction with normal and tumor cells by confocal microscopy;
vi) the assessment of the cytoxicity of pegylated MGL and MGL nanocapsules in the absence and presence of targeting peptides and antibodies in vivo;
vii) the determination of the pharmacokinetic properties of pegylated MGL and MGL nanocapsules in the bloodstream measuring the average retention time, the half-time distribution, the half-time of release.

Research goals

The project objective is the development of a novel drug for cancer therapy based on engineered methionine gamma lyase in order to deplete in methionine the medium of cancer cells that are defective in enzymes for methionine de novo and savage pathways. The specific objectives are:
1. to obtain mutant forms of MGL with higher catalytic efficiency in the reaction of gamma-elimination and higher stability with respect to the wild type enzyme;
2. to determine the structural basis ensuring high catalytic efficiency of the mutant form(s);
3. to obtain pegylated MGL mutants and MGL nanocapsules
4. to decorate pegylated MGL mutants and MGL nanocapsules with peptides and antibodies targeting cancer cells
5. to evaluate the cytotoxic activity of decorated and undecorated MGL nanocapsules and pegylated MGL on a panel of tumor cells;
6. to investigate the efficiency of binding of pegylated MGL and MGL nanocapsules to normal and tumor cells by confocal microscopy;
7. to evaluated the clinical effectiveness of MGL nanocapsules and pegylated MGL in vivo;
8. to determine the pharmacokinetic properties of MGL nanocapsules and pegylated MGL.

Last update: 28/03/2024