Anticandidal Activity of In Situ Methionine γ-Lyase-Based Thiosulfinate Generation System vs. Synthetic Thiosulfinates.

Candida albicans and non-albicans Candida species are a common cause of human mucosal infections, as well as bloodstream infections and deep mycoses. The emergence of resistance of Candida spp. to antifungal drugs used in practice requires the search for new antimycotics. The present study unravels the antifungal potential of the synthetic dialk(en)ylthiosulfinates in comparison with an enzymatic in situ methionine γ-lyase-based thiosulfinate generation system (TGS). The kinetics of the TGS reaction, namely, the methionine γ-lyase-catalyzed ß-elimination of S-alk(en)yl-L-cysteine sulfoxides, was investigated via 1H NMR spectroscopy for the first time, revealing fast conversion rates and the efficient production of anticandidal dialk(en)ylthiosulfinates. The anticandidal potential of this system vs. synthetic thiosulfinates was investigated through an in vitro assay. TGS proved to be more effective (MIC range 0.36-1.1 µg/mL) than individual substances (MIC range 0.69-3.31 µg/mL). The tested preparations had an additive effect with the commercial antimycotics fluconazole, amphotericin B and 5-flucytosine demonstrating a fractional inhibitory coefficient index in the range of 0.5-2 µg/mL. TGS can be regarded as an attractive candidate for the targeted delivery of antimycotic thiosulfinates and for further implementation onto medically implanted devices.

O-Acetylhomoserine Sulfhydrylase from Clostridioides difficile: Role of Tyrosine Residues in the Active Site.

O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5'-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.

Characteristics and Stability Assessment of Therapeutic Methionine γ-lyase-Loaded Polyionic Vesicles.

Pyridoxal 5'-phosphate-dependent methionine γ-lyase from Citrobacter freundii (MGL, EC 4.4.1.11) is studied as an antitumor enzyme and in combination with substrates as an antibacterial agent in enzyme pro-drug therapy. For the possibility of in vivo trials, two mutant forms, C115H MGL and V358Y MGL, were encapsulated into polyionic vesicles (PICsomes). Five pairs of polymers with the number of polymer chain units 20, 50, 70, 120, and 160 were synthesized. The effect of polymer length-PEGylated poly-l-aspartic acid and poly-l-lysine-on the degree of MGL incorporation into PICsomes and their size was investigated. Encapsulation of proteins in PICsomes is a rather new technique. Our data demonstrated that the length of the polymers and, therefore, the ratio of the hydrophobic and hydrophilic fragments most likely should be selected individually for each protein to be encapsulated. The efficiency of encapsulation of MGL mutant forms into PICsomes was up to 11%. The hydrodynamic diameter and surface potential of hollow and MGL-loaded PICsomes were evaluated by the dynamic light scattering method. The size and morphology of the PICsomes were determined by atomic force microscopy. The most acceptable for further in vivo studies were PICsomes20 with a size of 57-64 nm, PICsomes70 of 50-90 nm, and PICsomes120 of 100-105 nm. The analysis of the steady-state parameters has demonstrated that both mutant forms retained their catalytic properties inside the nanoparticles. The release study of the enzymes from PICsomes revealed that about 50% of the enzymes remained encapsulated in PICsomes70 and PICsomes120 after 24 h. Based on the data obtained, the most promising for in vivo studies are PICsomes70 and PICsomes120.

Kinetic and pharmacokinetic characteristics of therapeutic methinoninе γ-lyase encapsulated in polyion complex vesicles.

Therapeutic enzymes used for the treatment of a wide range of human disorders often suffer from suboptimal pharmacokinetics and stability. Engineering approaches such as encapsulation in micro- and nanocarriers, and replacements of amino acid residues of the native enzyme provide significant potential for improving the performance of enzyme therapy. Here, we develop a nanodelivery system on the base of polyion complex vesicles (PICsomes) that includes methionine γ-lyase (MGL) as a therapeutic enzyme. We have two strategies for using the enzyme: first, methionine γ-lyase is an anticancer agent removing l-methionine from plasma, second, the binary system methionine γ-lyase/S-alk(en)yl-l-cysteine sulfoxides is effective in enzyme prodrug therapy (EPT). Various lengths polymers were synthesized, and two mutant forms of the enzyme were used. The catalytic and pharmacokinetic parameters of the nanoformulations were investigated. The catalytic efficiencies of encapsulated enzymes were comparable to that of native enzymes. Pharmacokinetic analysis has shown that inclusion into PICsomes increases half-life of the enzymes, and they can be safely administered in vivo. The results suggest the further use of encapsulated MGLs for EPT and anticancer therapy, and this strategy could be leveraged to improve the efficiency of enzyme-based therapies for managing serious human diseases.