Sulfoxides, Analogues of L-Methionine and L-Cysteine As Pro-Drugs against Gram-Positive and Gram-Negative Bacteria

Abstract

The problem of resistance to antibiotics requires the development of new classes of broad-spectrum antimicrobial drugs. The concept of pro-drugs allows researchers to look for new approaches to obtain effective drugs with improved pharmacokinetic and pharmacodynamic properties. Thiosulfinates, formed enzymatically from amino acid sulfoxides upon crushing cells of genus Allium plants, are known as antimicrobial compounds. The instability and high reactivity of thiosulfinates complicate their use as individual antimicrobial compounds. We propose a pharmacologically complementary pair: an amino acid sulfoxide pro-drug and vitamin B6 – dependent methionine γ-lyase, which metabolizes it in the patient’s body. The enzyme catalyzes the γ- and β-elimination reactions of sulfoxides, analogues of L-methionine and L-cysteine, which leads to the formation of thiosulfinates. In the present work, we cloned the enzyme gene from Clostridium sporogenes. Ionic and tautomeric forms of the internal aldimine were determined by lognormal deconvolution of the holoenzyme spectrum and the catalytic parameters of the recombinant enzyme in the γ- and β-elimination reactions of amino acids, and some sulfoxides of amino acids were obtained. For the first time, the possibility of usage of the enzyme for effective conversion of sulfoxides was established and the antimicrobial activity of thiosulfinates against Gram-negative and Gram-positive bacteria in situ was shown.

Mutant formC115HofClostridium sporogenesmethionine γ‐lyase efficiently cleavesS‐Alk(en)yl‐l‐cysteine sulfoxides to antibacterial thiosulfinates

“…The Cys115/His mutant form was prepared as follows: the mgl‐sp‐Blue plasmid was obtained by cloning the megL_sporog gene into the Bluescript II vector SK(±); the mutagenesis was performed by PCR. The fragment containing T/C (343) and G/A (344) substitutions was amplified using mgl‐sp‐Blue as the template by PCR with the primers F1, F2, R1, and R2: …”

“…Recently, we have showed that PLP‐dependent MGL (EC 4.4.1.11) from Citrobacter freundii catalyzes the β‐elimination reaction of alliin and other S‐substituted l ‐cysteine sulfoxides to form allicin or S‐alkylthiosulfinates:

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“…Thiosulfinates in situ generated by the MGL/sulfoxide couple were demonstrated to possess antibacterial activity against some Gram‐positive and Gram‐negative bacteria . The main active principle of allicin and other thiosulfinates is the oxidation of SH groups of the proteins .…”

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

“…The band at 500 nm is typical of quinonoid intermediates that occur in reactions catalyzed by PLP‐dependent enzymes . The absorption spectrum of MGLs from C. sporogenes and C. tetani contains a band with a maximum at 505 nm , while this band is barely noticeable in the spectrum of MGL from C. freundii . The spectrum was resolved into bands corresponding to individual electron transitions.…”

“…Recently, we provided evidence for the cytotoxicity of MGL from C. sporogenes, C. tetani, and Citrobacter freundii MGL using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines (9). We also reported the capability of MGL to catalyze the formation of the potent antibacterial agent allicin or Salkylthiosulfinates from substrate analogues, such as alliin and S-substituted L-cysteine sulfoxides (10,11).…”