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Names | |
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IUPAC name 5-(2-Hydroxyethyl)-4-methyl-3-[(2-methyl-4-oxo-1,4-dihydropyrimidin-5-yl)methyl]-1,3-thiazol-3-ium | |
Other names Oxythiamine, 3-[(2-Methyl-4-oxo-1,4-dihydropyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium | |
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Properties | |
C12H16N3O2S+ (base); C12H17ClN3O2S (HCl) | |
Molar mass | 266.34 g/mol (base) 301.79 g/mol (HCl) |
Highly soluble in water (hydrochloride) | |
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). |
Oxythiamine (also known as OT) is a chemical analog of vitamin B1 (thiamine) and is classified as a thiamine antagonist. In the body, it is converted into its active form, oxythiamine pyrophosphate (OTP), which binds to thiamine-dependent enzymes and inhibits their function. [1]
Oxythiamine differs from thiamine in the structure of its pyrimidine ring: the amino group at position 6 is replaced with a keto group, forming pyrimidin-6-one. This alteration modifies the electronic properties of the molecule, affecting its reactivity.
Like thiamine, oxythiamine has a positively charged thiazolium cation, which is neutralized by a chloride ion. The hydroxyethyl side chain, which is important for its function, and the methyl group on the thiazole ring are retained. This structural similarity explains why oxythiamine can bind to the same enzymes as thiamine.
In the body, thiamine is converted into thiamine pyrophosphate (TPP), an essential coenzyme. TPP plays a critical role in enzymatic reactions by stabilizing reactive intermediates – particularly carbanions – in thiamine-dependent enzymes such as transketolase.
Oxythiamine is also phosphorylated by the enzyme thiamine pyrophokinase, producing oxythiamine pyrophosphate (OTP). While OTP can bind to the same enzymes as TPP, it is unable to stabilize carbanions. This is due to the keto group in OTP, which disrupts electron delocalization in the thiazolium ring. As a result, OTP acts as a competitive inhibitor, blocking enzyme activity without enabling catalysis. [1]
One of OTP's most important targets is transketolase, a key enzyme in the pentose phosphate pathway (PPP). Inhibiting transketolase halts the non-oxidative branch of the PPP, which severely limits the formation of ribose-5-phosphate—a critical precursor for nucleotide synthesis. This inhibition also impairs the generation of NADPH, an essential reducing agent in biosynthesis and antioxidant defense.
Because both nucleotides and NADPH are indispensable for cell growth and division, particularly in rapidly proliferating tumor cells, OTP exerts a selective anti-proliferative effect on such cells.
Oxythiamine is enzymatically converted into its active form, oxythiamine pyrophosphate (OTP), in a variety of organisms – including bacteria, protozoan parasites, and mammalian cells—through phosphorylation by the enzyme thiamine pyrophokinase. [2] Structurally, OTP closely resembles the natural coenzyme thiamine diphosphate (ThDP or TPP), sharing key functional features that allow it to bind to the same enzymatic sites.
However, unlike ThDP, OTP is catalytically inactive: it fails to support the necessary stabilization of reaction intermediates, thereby preventing normal enzymatic function. By occupying ThDP binding sites without contributing to catalysis, OTP effectively acts as a metabolic decoy, shutting down thiamine-dependent processes.
A pivotal study by Chan et al. demonstrated that overexpression of thiamine pyrophokinase in Plasmodium falciparum – the parasite responsible for malaria – resulted in a 1,700-fold increase in sensitivity to oxythiamine. [2] This strongly indicates that the cytotoxic and antiproliferative effects of oxythiamine are not due to the parent compound itself, but rather to its phosphorylated metabolite, OTP, which functions as the true active antimetabolite.
Oxythiamine (OT) and its active metabolite, oxythiamine pyrophosphate (OTP), inhibit several key enzymes that depend on thiamine diphosphate (ThDP, also known as TPP) as a cofactor. These ThDP-dependent enzymes are critical for central metabolic pathways and include:
Transketolase requires ThDP to perform its catalytic function. Oxythiamine pyrophosphate (OTP) competes for the same cofactor binding site, acting as a competitive inhibitor. In cell culture experiments with mammalian and cancer cell lines, oxythiamine significantly reduced transketolase activity, limiting the production of ribose-5-phosphate, a precursor for nucleotide biosynthesis. [3]
This effect is particularly pronounced in rapidly dividing cells, such as tumor cells, which experience a shortage of nucleotide building blocks. This can lead to cell cycle arrest in the G1 phase and initiate apoptosis. [2] Moreover, OTP binds transketolase with high affinity and shows limited reversibility under physiological conditions. [4] [5]
Importantly, OTP also inhibits transketolase isoforms such as TKTL1, which are frequently overexpressed in certain cancers and have been implicated in tumor progression and metastasis. [6]
The active coenzyme thiamine diphosphate (ThDP) binds with particularly high affinity to human transketolase (TKT). Structural studies have shown that specific amino acids – such as glutamine at position 189 (Gln189) – are essential for cofactor stabilization. Additional stabilizing interactions, involving residues such as Lys75, Ser40, and Lys244, contribute to the near-irreversible binding of ThDP under physiological conditions. [7]
Because oxythiamine pyrophosphate (OTP) cannot easily displace this strongly bound ThDP, oxythiamine primarily acts as a preventive inhibitor. It competes with thiamine for incorporation into newly synthesized enzymes. This results in enzyme variants that are inactive from the outset.
The pyruvate dehydrogenase complex is a mitochondrial enzyme complex that catalyzes the conversion of pyruvate to acetyl-CoA, serving as a critical link between glycolysis and the citrate cycle. Its E1 subunit requires ThDP as a cofactor. OTP acts as a competitive inhibitor at this site and even exhibits higher binding affinity than ThDP itself: its inhibition constant (Ki) is approximately 0.025 μM, compared to a Km of ~0.06 μM for ThDP. [4]
When PDHC is inhibited by OTP, intracellular pyruvate accumulates and is increasingly diverted to lactate production. Concurrently, acetyl-CoA synthesis declines, reducing input into the citrate cycle and impairing energy metabolism. In high-glucose-demand tissues—such as tumors or rapidly dividing microbes—this disruption can have severe metabolic consequences. In rat models, oxythiamine administration significantly increased blood pyruvate levels, confirming its systemic inhibitory effect on PDHC. [8]
The 2-oxoglutarate dehydrogenase complex (OGDHC) is another ThDP-dependent enzyme in the citrate cycle. Like PDHC, it is effectively inhibited by oxythiamine pyrophosphate (OTP). Experimental studies have shown that parasites genetically engineered to overexpress OGDHC exhibit reduced sensitivity to oxythiamine – suggesting that excess enzyme can sequester available OTP, thereby preserving partial enzymatic activity. [2]
In tumor cells, oxythiamine treatment has been shown to cause an accumulation of α-ketoglutarate, the substrate of OGDHC. [6] This metabolite plays a key role as a signaling molecule, influencing both epigenetic enzymes and the hypoxia-inducible factor HIF-1α. Elevated α-ketoglutarate levels may promote HIF hydroxylase stabilization, thereby reducing HIF-1α activity—a mechanism with potential anti-metastatic implications.
OTP can bind to enzymes that normally rely on thiamine diphosphate (ThDP). Due to its structural similarity, it competes with the natural cofactor for the binding sites on these enzymes. [4] However, unlike ThDP, OTP lacks the electronic properties necessary to stabilize reactive intermediates and therefore fails to support enzymatic catalysis. The result is a functional blockade of metabolic reactions dependent on these enzymes.
Importantly, oxythiamine's inhibitory effect is not limited to direct competition. Once phosphorylated to OTP, it induces a persistent inhibition that continues until sufficient thiamine is available to regenerate active ThDP. Cell culture experiments have demonstrated that high doses of thiamine can partially reverse this inhibition, underscoring its competitive and reversible nature. [4]
However, in many cases, enzyme activity is not fully restored, suggesting that OTP may bind with high affinity or induce structural alterations that lead to long-term enzymatic dysfunction.
Inhibition of ThDP-dependent enzymes by oxythiamine pyrophosphate (OTP) disrupts core metabolic pathways, leading to wide-ranging biochemical consequences:
![]() | This article may contain an excessive amount of intricate detail that may only interest a particular audience.(May 2025) |
Oxythiamine is primarily used as a research compound to induce cellular thiamine deficiency in a controlled, selective manner. It serves as a valuable tool in studies of cell metabolism, especially within the fields of oncology, microbiology, parasitology, and immunology.
Oxythiamine has been investigated in numerous in vitro studies with regard to its tumor-inhibiting properties. It inhibits thiamine-dependent enzymes, interferes with cell proliferation, induces apoptosis and influences the cell cycle. The inhibition primarily affects transketolase (TKT) and its isoforms such as TKTL1.
Clinical data are lacking. Old animal studies showed virus inhibition, but also immunological weakening. BOT is discussed as a nasal or inhaled antiviral therapy. [31]
Case report: Prostate cancer and benfo-oxythiamine. A case report from 2024 describes the combined use of benfo-oxythiamine (B-OT) and [177Lu]PSMA radioligand therapy in a patient with metastatic, refractory prostate cancer. The therapy resulted in:
There was no evidence of systemic thiamine deficiency or other relevant side effects. [38]
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