Thiamine and selected thiamine antivitamins - biological activity and methods of synthesis.

Thiamine plays a very important coenzymatic and non-coenzymatic role in the regulation of basic metabolism. Thiamine diphosphate is a coenzyme of many enzymes, most of which occur in prokaryotes. Pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes as well as transketolase are the examples of thiamine-dependent enzymes present in eukaryotes, including human. Therefore, thiamine is considered as drug or diet supplement which can support the treatment of many pathologies including neurodegenerative and vascular system diseases. On the other hand, thiamine antivitamins, which can interact with thiamine-dependent enzymes impeding their native functions, thiamine transport into the cells or a thiamine diphosphate synthesis, are good propose to drug design. The development of organic chemistry in the last century allowed the synthesis of various thiamine antimetabolites such as amprolium, pyrithiamine, oxythiamine, or 3-deazathiamine. Results of biochemical and theoretical chemistry research show that affinity to thiamine diphosphate-dependent enzymes of these synthetic molecules exceeds the affinity of native coenzyme. Therefore, some of them have already been used in the treatment of coccidiosis (amprolium), other are extensively studied as cytostatics in the treatment of cancer or fungal infections (oxythiamine and pyrithiamine). This review summarizes the current knowledge concerning the synthesis and mechanisms of action of selected thiamine antivitamins and indicates the potential of their practical use.

Involvement of thiaminase II encoded by the THI20 gene in thiamin salvage of Saccharomyces cerevisiae.

The physiological significance of thiaminase II, which catalyzes the hydrolysis of thiamin, has remained elusive for several decades. The C-terminal domains of THI20 family proteins (THI20/21/22) and the whole region of PET18 gene product of Saccharomyces cerevisiae are homologous to bacterial thiaminase II. On the other hand, the N-terminal domains of THI20 and THI21 encode 2-methyl-4-amino-5-hydroxymethylpyrimidine kinase and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase involved in the thiamin synthetic pathway. In this study, it was first indicated that the C-terminal domains of the THI20 family and PET18 are not required for de novo thiamin synthesis in S. cerevisiae, using a quadruple deletion strain expressing the N-terminal domain of THI20. Biochemical analysis using cell-free extracts and recombinant proteins demonstrated that yeast thiaminase II activity is exclusively encoded by THI20. It appeared that Thi20p has an affinity for the pyrimidine moiety of thiamin, and HMP produced by the thiaminase II activity is immediately phosphorylated. Thi20p was found to participate in the formation of thiamin from two synthetic antagonists, pyrithiamin and oxythiamin, by hydrolyzing both antagonists and phosphorylating HMP to give HMP pyrophosphate. Furthermore, 2-methyl-4-amino-5-aminomethylpyrimidine, a presumed naturally occurring thiamin precursor, was effectively converted to HMP by incubation with Thi20p. It is proposed that the thiaminase II activity of Thi20p is involved in the thiamin salvage pathway by catalyzing the hydrolysis of HMP precursors in S. cerevisiae.

Pyrithiamine as a substrate for thiamine pyrophosphokinase.

Thiamine pyrophosphokinase transfers a pyrophosphate group from a nucleoside triphosphate, such as ATP, to the hydroxyl group of thiamine to produce thiamine pyrophosphate. Deficiencies in thiamine can result in the development of the neurological disorder Wernicke-Korsakoff Syndrome as well as the potentially fatal cardiovascular disease wet beriberi. Pyrithiamine is an inhibitor of thiamine metabolism that induces neurological symptoms similar to that of Wernicke-Korsakoff Syndrome in animals. However, the mechanism by which pyrithiamine interferes with cellular thiamine phosphoester homeostasis is not entirely clear. We used kinetic assays coupled with mass spectrometry of the reaction products and x-ray crystallography of an equilibrium reaction mixture of thiamine pyrophosphokinase, pyrithiamine, and Mg2+/ATP to elucidate the mechanism by which pyrithiamine inhibits the enzymatic production of thiamine pyrophosphate. Three lines of evidence support the ability of thiamine pyrophosphokinase to form pyrithiamine pyrophosphate. First, a coupled enzyme assay clearly demonstrated the ability of thiamine pyrophosphokinase to produce AMP when pyrithiamine was used as substrate. Second, an analysis of the reaction mixture by mass spectrometry directly identified pyrithiamine pyrophosphate in the reaction mixture. Last, the structure of thiamine pyrophosphokinase crystallized from an equilibrium substrate/product mixture shows clear electron density for pyrithiamine pyrophosphate bound in the enzyme active site. This structure also provides the first clear picture of the binding pocket for the nucleoside triphosphate and permits the first detailed understanding of the catalytic requirements for catalysis in this enzyme.

Dual system of intestinal thiamine transport in humans.

The transport of thiamine across the intestine has been characterized in rats but has not been adequately studied in humans. To determine the kinetics of thiamine intestinal transport directly in humans, mucosal tissues were obtained during routine endoscopy from normal-appearing sites at the second portion of the duodenum. With 3H-dextran as the marker of adherent volume, the uptake of 14C-thiamine hydrochloride by the excised mucosa was measured in vitro. By this method thiamine uptake was linear with tissue weight and with incubation time up to 5 min. Results showed that at low thiamine concentrations (0.2 to 2.0 microM), uptake was saturable whereas at high concentrations (5 to 50 microM), uptake was linear with thiamine concentrations. Pyrithiamine, anoxia, N-ethylmaleimide, and replacement of sodium chloride by mannitol reduced the uptake of 0.5 microM thiamine by 42%, 37%, 32% and 35%, respectively (p less than 0.05) but had no effect on the uptake of 20 microM thiamine. These data suggest that, as in the rat, the intestinal transport of thiamine in humans proceeds by a coexistent dual system. At physiologic concentrations, thiamine is transported primarily by an energy-requiring, sodium-dependent active process, whereas at higher pharmacologic concentrations thiamine uptake is predominantly a passive process.

[Effects of oxythiamine and pyrithiamine on rat liver. --Biochemical and morphological changes in the thiamine deficient rat (author's transl)].

We studied biochemical changes in the rat liver in a thiamine (T) deficient state as induced by oxythiamine (OT), pyrithiamine (PT) and thiamine deficient diet (TDD) and simultaneously observed morphological changes under light and electron microscope. Severe loss of weight was observed in the OT treated rats fed on TDD (OTD) and such was frequently accompanied by a complete loss of righting reflex. Biochemical changes commonly found in T deficient groups were decrease in serum total protein, alkaline phosphatase activity and liver lipids, and increase in serum total cholesterol and transaminase activity. Microscopically, most of the liver cells were atrophied and necrosis was observed in the OTD group. Electron microscopically, ultrastructural changes revealed active Kupffer cells, microvilli and Golgi apparatus, and decrease in rough endoplasmic reticulum (rER) associated with increasing detached ribosomes and smooth ER. Abnormal nucleus and mitochondria were found in the OTD group. These results suggest that a T deficiency occurs readily and easily within a short time when a T antagonist, particularly OT, is used together with the TDD, while a much longer time is required to produce a T deficiency with only the TDD.

Inhibition of thiamine transport in Saccharomyces cerevisiae by thiamine disulfides.

Both thiamine disulfide and O-benzoyl thiamine disulfide, which are thiolfrom derivatives of thiamine, strongly inhibited thiamine transport in Saccharomyces cerevisiae. The inhibition appeared to be due to a high affinity of the analogs for yeast cell membranes, in which thiamine transport component(s) may be integrated.