Thiosulfinates: Cytotoxic and Antitumor Activity.

Pharmacological value of some natural compounds makes them attractive for use in oncology. The sulfur-containing thiosulfinates found in plants of the genus Allium have long been known as compounds with various therapeutic properties, including antitumor. Over the last few years, the effect of thiosulfinates on various stages of carcinogenesis has been actively investigated. In vitro and in vivo studies have shown that thiosulfinates inhibit proliferation of cancer cells, as well as they induce apoptosis. The purpose of this review is to summarize current data on the use of natural and synthetic thiosulfinates in cancer therapy. Antitumor mechanisms and molecular targets of these promising compounds are discussed. A significant part of the review is devoted to consideration of a new strategy for treatment of oncological diseases - use of the directed enzyme prodrug therapy approach aiming to obtain antitumor thiosulfinates in situ.

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.

Novel Hydroxamic Acids Containing Aryl-Substituted 1,2,4- or 1,3,4-Oxadiazole Backbones and an Investigation of Their Antibiotic Potentiation Activity.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc amidase that catalyzes the second step of the biosynthesis of lipid A, which is an outer membrane essential structural component of Gram-negative bacteria. Inhibitors of this enzyme can be attributed to two main categories, non-hydroxamate and hydroxamate inhibitors, with the latter being the most effective given the chelation of Zn2+ in the active site. Compounds containing diacetylene or acetylene tails and the sulfonic head, as well as oxazoline derivatives of hydroxamic acids, are among the LpxC inhibitors with the most profound antibacterial activity. The present article describes the synthesis of novel functional derivatives of hydroxamic acids-bioisosteric to oxazoline inhibitors-containing 1,2,4- and 1,3,4-oxadiazole cores and studies of their cytotoxicity, antibacterial activity, and antibiotic potentiation. Some of the hydroxamic acids we obtained (9c, 9d, 23a, 23c, 30b, 36) showed significant potentiation in nalidixic acid, rifampicin, and kanamycin against the growth of laboratory-strain Escherichia coli MG1655. Two lead compounds (9c, 9d) significantly reduced Pseudomonas aeruginosa ATCC 27853 growth in the presence of nalidixic acid and rifampicin.

O-acetylhomoserine sulfhydrylase from Clostridium novyi. Cloning, expression of the gene and characterization of the enzyme.

The gene NT01CX_1210 of pathogenic bacterium Clostridium novyi annotated as encoding O-acetylhomoserine sulfhydrylase was cloned and expressed in Escherichia coli. The gene product having O-acetylhomoserine sulfhydrylase activity was purified to homogeneity. The protein showed molecular mass of approximately 184 kDa for the native form and 46 kDa for the subunit. The enzyme catalyzes the γ-substitution reaction of O-acetylhomoserine with maximum activity at pH 7.5. Analysis of C. novyi genome allowed us to suggest that there is only one way for the synthesis of l-methionine in the bacterium. The data obtained may provide the basis for further study of the role of OAHS in Clostridium bacteria and an ascertainment of its mechanism.

Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations.

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

Soluble and Nanoporous Silica Gel-Entrapped C. freundii Methionine γ-Lyase.

Methionine γ-lyase is a pyridoxal 5'-phosphate dependent tetramer that catalyzes the α,γ-elimination of methionine in ammonia, methanethiol and α-ketobutyrate. MGL catalytic power has been exploited as a therapeutic strategy to reduce the viability of cancer cells or bacteria. In order to obtain a stable enzyme to be delivered at the site of action, MGL can be encapsulated in a variety of matrices. As a reference encapsulation strategy we have prepared MGL nanoporous wet silica gels. Immobilized MGL gels were characterized with regards to activity, stability, absorption, circular dichroism and fluorescence properties and compared with soluble MGL. We found that MGL gels exhibit (i) spectroscopic properties very similar to MGL in solution, (ii) a higher stability with respect to the soluble enzyme and (iii) catalytic activity six-fold lower than in solution. These findings prove that MGL encapsulation is a suitable strategy for therapeutic applications.

Gene cloning, characterization, and cytotoxic activity of methionine γ-lyase from Clostridium novyi.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits kcat and kcat /Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL-1 , 4.4 U mL-1 , 1.2 U mL-1 , and 3.4 U mL-1 , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017.

Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with l-norleucine.

The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-l-homoserine and in the ß-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and ß-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.

Pre-steady-state kinetic and structural analysis of interaction of methionine γ-lyase from Citrobacter freundii with inhibitors.

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the ß-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.

The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and ß-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-ß- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and ß-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the ß-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016.

Alliin is a suicide substrate of Citrobacter freundii methionine γ-lyase: structural bases of inactivation of the enzyme.

The interaction of Citrobacter freundii methionine γ-lyase (MGL) and the mutant form in which Cys115 is replaced by Ala (MGL C115A) with the nonprotein amino acid (2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) was investigated. It was found that MGL catalyzes the ß-elimination reaction of alliin to form 2-propenethiosulfinate (allicin), pyruvate and ammonia. The ß-elimination reaction of alliin is followed by the inactivation and modification of SH groups of the wild-type and mutant enzymes. Three-dimensional structures of inactivated wild-type MGL (iMGL wild type) and a C115A mutant form (iMGL C115A) were determined at 1.85 and 1.45 Šresolution and allowed the identification of the SH groups that were oxidized by allicin. On this basis, the mechanism of the inactivation of MGL by alliin, a new suicide substrate of MGL, is proposed.

Exploring methionine γ-lyase structure-function relationship via microspectrophotometry and X-ray crystallography.

Pyridoxal 5'-phosphate (PLP) dependent methionine γ-lyase catalyzes the breakdown of L-methionine to α-ketobutyric acid, methanethiol and ammonia. This enzyme, present in anaerobic microorganisms, has biomedical interest both for its activity as antitumor agent, depleting methionine supply in methionine-dependent cancers, and as target in the treatment of human pathogen infections, activating the pro-drug trifluoromethionine. To validate the structure of the enzyme from Citrobacter freundii, crystallized from monomethyl ether polyethylene glycol 2000, for the development of lead compounds, the reactivity of the crystalline enzyme towards L-methionine, substrate analogs and inhibitors was determined by polarized absorption microspectrophotometry. Spectral data were also collected for enzyme crystals, grown in monomethyl ether polyethylene glycol 2000 in the presence of ammonium sulfate. The three-dimensional structure of these enzyme crystals, solved at 1.65Å resolution with R(free) 23.2%, revealed the surprising absence of the aldimine bond between the active site Lys210 and PLP. Different hypothesis are proposed and discussed in the light of spectral and structural data, pointing out to the relevance of the complementarity between X-ray crystallography and single crystal spectroscopy for the understanding of biological mechanisms at molecular level. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

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.

Methionine gamma-lyase: mechanistic deductions from the kinetic pH-effects. The role of the ionic state of a substrate in the enzymatic activity.

We have studied and compared the pH-dependencies of the main kinetic parameters for the alpha,gamma-elimination reactions of methionine gamma-lyase (MGL) of Citrobacter intermedius with natural substrate, l-methionine, with its phosphinic analogue, and for alpha,beta-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k(cat)/K(m) for the reaction with l-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the pK(a) of the substrate's amino group, equal to 7.55, is not reflected in the pH-profile of k(cat)/K(m). Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k(cat)/K(m) for reactions with l-methionine and S-methyl-l-cysteine are controlled by a single pK(a) equal to 7.1-7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal pK(a)s of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k(cat)/K(m) for the reactions with S-methyl-l-cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average pK(a) values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both alpha,gamma- and alpha,beta-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the alpha,gamma-elimination, and Tyr 58 - in the alpha,beta-elimination process.

Methionine γ-lyase in enzyme prodrug therapy: An improvement of pharmacokinetic parameters of the enzyme.

Citrobacter freundii methionine γ-lyase (MGL), in addition to the physiological reaction, catalyzes the ß-elimination reaction of S-alk(en)yl-L-cysteine sulfoxides to yield thiosulfinates, which have antibacterial activity. We have obtained the mutant form C115H MGL, which cleaves S-alk(en)yl-L-cysteine sulfoxides more effectively than the wild type enzyme does. The binary system MGL/S-alk(en)yl-L-cysteine sulfoxides may be considered as a new pharmacological pair in enzyme prodrug therapy (EPT). Despite of the successful application of this pair in antibacterial studies in vitro, in vivo experiments may lead to several problems typical of therapeutic proteins including a relatively short-lasting biological activity. To circumvent these problems, we have investigated several approaches to improve safety and efficacy of the enzyme component of the pharmacological pair. This included covalent attachment of poly(ethylene glycol) to the enzyme, its encapsulation in liposomes and polymeric vesicles (PICsomes). The steady-state and pharmacokinetic parameters of modified/encapsulated enzyme were determined. It was demonstrated that the encapsulation in PICsomes prolongs in vivo stability of C115H MGL to over 42 h compared to PEGylated enzyme (3 h). Antibacterial activity of binary system ("pharmacological pair") modified/encapsulated enzyme/S-alk(en)yl-L-cysteine sulfoxides was tested and remained the same as for the naked enzyme. Thus, the usage of MGL-loaded PICsomes as enzymatic nanoreactors in ETP to produce antimicrobial thiosulfinates is promising.

Serine 51 residue of Citrobacter freundii tyrosine phenol-lyase assists in C-α-proton abstraction and transfer in the reaction with substrate.

In the spatial structure of tyrosine phenol-lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l-tyrosine and 3-fluoro-l-tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4'-position of the leaving phenol group in the reaction with the natural substrate.

Engineered Citrobacter freundii methionine γ-lyase effectively produces antimicrobial thiosulfinates.

Antimicrobial activity of thiosulfinates in situ produced by mixtures of Citrobacter freundii methionine γ-lyase (MGL) with new substrates, l-methionine and S-(alkyl/allyl)-l-cysteine sulfoxides has been recently demonstrated (Anufrieva et al., 2015). This opens a way to the rational design of a new biotechnologically relevant antimicrobial drug producer. To increase the efficiency of the enzyme toward sulfoxides, the mutant forms of MGL, with the replacements of active site cysteine 115 with alanine (C115A MGL) and histidine (C115H MGL) were obtained. The replacement of cysteine 115 by histidine results in the loss of activity of the mutant enzyme in the γ-elimination reaction of physiological substrate, whereas the activity in the ß-elimination reaction of characteristic substrates persists. However, the catalytic efficiency of C115H MGL in the ß-elimination reaction of S-substituted l-cysteine sulfoxides is increased by about an order of magnitude compared to the wild type MGL. The antibacterial activity of C115H MGL mixtures with a number of sulfoxides was assessed against Gram-positive and Gram-negative bacteria. The bacteriostatic effect was more pronounced against Gram-positive than against Gram-negative bacteria, while antibacterial potential proved to be quite similar. Thus, the mutant enzyme C115H MGL is an effective catalyst, in particular, for decomposition of sulfoxides and the pharmacological couples of the mutant form with sulfoxides might be new antimicrobial agents.

Crystal structure of the external aldimine of Citrobacter freundii methionine γ-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of α- and ß-protons of competitive inhibitors.

The three-dimensional structure of the external aldimine of Citrobacter freundii methionine γ-lyase with competitive inhibitor glycine has been determined at 2.45 Å resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of Cα-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of Cα-proton to C4'-atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic Cα-protons of glycine may be explained on the basis of external aldimine structure. The exchange of Cα-pro-(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from Cα-atom of glycine to C4'-atom of the cofactor. The exchange of Cα-pro-(S)-proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of ß-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid.