Identification and characterization of thiamine analogs with antiplasmodial activity.

Thiamine is metabolized into thiamine pyrophosphate (TPP), an essential enzyme cofactor. Previous work has shown that oxythiamine, a thiamine analog, is metabolized by thiamine pyrophosphokinase (TPK) into oxythiamine pyrophosphate within the malaria parasite Plasmodium falciparum and then inhibits TPP-dependent enzymes, killing the parasite in vitro and in vivo. To identify a more potent antiplasmodial thiamine analog, 11 commercially available compounds were tested against P. falciparum and P. knowlesi. Five active compounds were identified, but only N3-pyridyl thiamine (N3PT), a potent transketolase inhibitor and candidate anticancer lead compound, was found to suppress P. falciparum proliferation with an IC50 value 10-fold lower than that of oxythiamine. N3PT was active against P. knowlesi and was >17 times less toxic to human fibroblasts, as compared to oxythiamine. Increasing the extracellular thiamine concentration reduced the antiplasmodial activity of N3PT, consistent with N3PT competing with thiamine/TPP. A transgenic P. falciparum line overexpressing TPK was found to be hypersensitized to N3PT. Docking studies showed an almost identical binding mode in TPK between thiamine and N3PT. Furthermore, we show that [3H]thiamine accumulation, resulting from a combination of transport and metabolism, in isolated parasites is reduced by N3PT. Treatment of P. berghei-infected mice with 200 mg/kg/day N3PT reduced their parasitemia, prolonged their time to malaria symptoms, and appeared to be non-toxic to mice. Collectively, our studies are consistent with N3PT competing with thiamine for TPK binding and inhibiting parasite proliferation by reducing TPP production, and/or being converted into a TPP antimetabolite that inhibits TPP-dependent enzymes.

Thiamine analogues featuring amino-oxetanes as potent and selective inhibitors of pyruvate dehydrogenase.

Pyruvate dehydrogenase complex (PDHc) is suppressed in some cancer types but overexpressed in others. To understand its contrasting oncogenic roles, there is a need for selective PDHc inhibitors. Its E1-subunit (PDH E1) is a thiamine pyrophosphate (TPP)-dependent enzyme and catalyses the first and rate-limiting step of the complex. In a recent study, we reported a series of ester-based thiamine analogues as selective TPP-competitive PDH E1 inhibitors with low nanomolar affinity. However, when the ester linker was replaced with an amide for stability reasons, the binding affinity was significantly reduced. In this study, we show that an amino-oxetane bioisostere of the amide improves the affinity and maintains stability towards esterase-catalysed hydrolysis.

Open-chain thiamine analogues as potent inhibitors of thiamine pyrophosphate (TPP)-dependent enzymes.

A common approach to studying thiamine pyrophosphate (TPP)-dependent enzymes is by chemical inhibition with thiamine/TPP analogues which feature a neutral aromatic ring in place of the positive thiazolium ring of TPP. These are potent inhibitors but their preparation generally involves multiple synthetic steps to construct the central ring. We report efficient syntheses of novel, open-chain thiamine analogues which potently inhibit TPP-dependent enzymes and are predicted to share the same binding mode as TPP. We also report some open-chain analogues that inhibit pyruvate dehydrogenase E1-subunit (PDH E1) and are predicted to occupy additional pockets in the enzyme other than the TPP-binding pockets. This opens up new possibilities for increasing the affinity and selectivity of the analogues for PDH, which is an established anti-cancer target.

Furan-based inhibitors of pyruvate dehydrogenase: SAR study, biochemical evaluation and computational analysis.

Suppression of pyruvate dehydrogenase complex (PDHc) is a mechanism for cancer cells to manifest the Warburg effect. However, recent evidence suggests that whether PDHc activity is suppressed or activated depends on the type of cancer. The PDHc E1 subunit (PDH E1) is a thiamine pyrophosphate (TPP)-dependent enzyme, catalysing the first and rate-limiting step of PDHc; thus, there is a need for selective PDH E1 inhibitors. There is, however, inadequate understanding of the structure-activity relationship (SAR) and a lack of inhibitors specific for mammalian PDH E1. Our group have reported TPP analogues as TPP-competitive inhibitors to study the family of TPP-dependent enzymes. Most of these TPP analogues cannot be used to study PDHc in cells because (a) they inhibit all members of the family and (b) they are membrane-impermeable. Here we report derivatives of thiamine/TPP analogues that identify elements distinctive to PDH E1 for selectivity. Based on our SAR findings, we developed a series of furan-based thiamine analogues as potent, selective and membrane-permeable inhibitors of mammalian PDH E1. We envision that our SAR findings and inhibitors will aid work on using chemical inhibition to understand the oncogenic role of PDHc.

Design of thiamine analogues for inhibition of thiamine diphosphate (ThDP)-dependent enzymes: Systematic investigation through Scaffold-Hopping and C2-Functionalisation.

Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positively charged thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) which is the best aromatic ring? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can further improve both potency and selectivity.

Synthesis of pyrrothiamine, a novel thiamine analogue, and evaluation of derivatives as potent and selective inhibitors of pyruvate dehydrogenase.

Inhibition of thiamine pyrophosphate (TPP)-dependent enzymes with thiamine/TPP analogues that have the central thiazolium ring replaced with other rings is well established, but a limited number of central rings have been reported. We report a novel analogue, pyrrothiamine, with a central pyrrole ring. We further develop pyrrothiamine derivatives as potent and selective inhibitors of pyruvate dehydrogenase, which might have anti-cancer potential.

Triazole-Based Thiamine Analogues as Inhibitors of Thiamine Pyrophosphate-Dependent Enzymes: 1,3-Dicarboxylate for Metal Binding.

Thiamine 1 (vitamin B1) is essential for energy metabolism, and interruption of its utilization pathways is linked to various disease states. Thiamine pyrophosphate 2a (TPP, the bioactive form of 1) functions as a coenzyme of a variety of enzymes. To understand the role of vitamin B1 in these diseases, a chemical approach is to use coenzyme analogues to compete with TPP for the enzyme active site, which abolishes the coenzyme function. Exemplified by oxythiamine 3a and triazole hydroxamate 4, chemical probes require the coenzyme analogues to be membrane-permeable and of broad inhibitory activity to the enzyme family (rather than being too selective to particular TPP-dependent enzymes). In this study, using biochemical assays, we show that changing the hydroxamate metal-binding group of 4 to a 1,3-dicarboxylate moiety leads to the potent inhibition of multiple TPP-dependent enzymes. We further demonstrate that this dianionic thiamine analogue when masked in its diester form becomes membrane-permeable and can be unmasked by esterase treatment. Taken together, our inhibitors are potentially useful chemical tools to study the roles of vitamin B1, using a prodrug mechanism, to induce the effects of thiamine deficiency in cell-based assays.

Evaluation of ketoclomazone and its analogues as inhibitors of 1-deoxy-d-xylulose 5-phosphate synthases and other thiamine diphosphate (ThDP)-dependent enzymes.

Most pathogenic bacteria, apicomplexan parasites and plants rely on the methylerythritol phosphate (MEP) pathway to obtain precursors of isoprenoids. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS), a thiamine diphosphate (ThDP)-dependent enzyme, catalyses the first and rate-limiting step of the MEP pathway. Due to its absence in humans, DXPS is considered as an attractive target for the development of anti-infectious agents and herbicides. Ketoclomazone is one of the earliest reported inhibitors of DXPS and antibacterial and herbicidal activities have been documented. This study investigated the activity of ketoclomazone on DXPS from various species, as well as the broader ThDP-dependent enzyme family. To gain further insights into the inhibition, we have prepared analogues of ketoclomazone and evaluated their activity in biochemical and computational studies. Our findings support the potential of ketoclomazone as a selective antibacterial agent.

ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

Correction: Thiamine analogues as inhibitors of pyruvate dehydrogenase and discovery of a thiamine analogue with non-thiamine related antiplasmodial activity.

[This corrects the article DOI: 10.1039/D2MD00085G.].

Inhibition of Thiamine Diphosphate-Dependent Enzymes by Triazole-Based Thiamine Analogues.

Thiamine is metabolized into the coenzyme thiamine diphosphate (ThDP). Interrupting thiamine utilization leads to disease states. Oxythiamine, a thiamine analogue, is metabolized into oxythiamine diphosphate (OxThDP), which inhibits ThDP-dependent enzymes. Oxythiamine has been used to validate thiamine utilization as an anti-malarial drug target. However, high oxythiamine doses are needed in vivo because of its rapid clearance, and its potency decreases dramatically with thiamine levels. We report herein cell-permeable thiamine analogues possessing a triazole ring and a hydroxamate tail replacing the thiazolium ring and diphosphate groups of ThDP. We characterize their broad-spectrum competitive inhibition of ThDP-dependent enzymes and of Plasmodium falciparum proliferation. We demonstrate how the cellular thiamine-utilization pathway can be probed by using our compounds and oxythiamine in parallel.

Prodrugs of pyrophosphates and bisphosphonates: disguising phosphorus oxyanions.

Pyrophosphates have important functions in living systems and thus pyrophosphate-containing molecules and their more stable bisphosphonate analogues have the potential to be used as drugs for treating many diseases including cancer and viral infections. Both pyrophosphates and bisphosphonates are polyanionic at physiological pH and, whilst this is essential for their biological activity, it also limits their use as therapeutic agents. In particular, the high negative charge density of these compounds prohibits cell entry other than by endocytosis, prevents transcellular oral absorption and causes sequestration to bone. Therefore, prodrug strategies have been developed to temporarily disguise the charges of these compounds. This review examines the various systems that have been used to mask the phosphorus-containing moieties of pyrophosphates and bisphosphonates and also illustrates the utility of such prodrugs.

Thiamine analogues as inhibitors of pyruvate dehydrogenase and discovery of a thiamine analogue with non-thiamine related antiplasmodial activity.

A series of derivatives of a triazole analogue of thiamine has been synthesised. When tested as inhibitors of porcine pyruvate dehydrogenase, the benzoyl ester derivatives proved to be potent thiamine pyrophosphate (TPP) competitive inhibitors, with the affinity of the most potent analogue (K i = 54 nM) almost matching the affinity of TPP itself. When tested as antiplasmodials, most of the derivatives showed modest activity (IC50 value >60 µM), except for a 4'-N-benzyl derivative, which has an IC50 value in the low micromolar range. This activity was not affected by increasing the extracellular concentration of thiamine in the culture medium for any of the compounds (except a modest increase in the IC50 for the unfunctionalized benzoyl ester), nor by overexpressing thiamine pyrophosphokinase in the parasite, making it unlikely to be due to an effect on thiamine transport or metabolism.

Thirty Years of HDAC Inhibitors: 2020 Insight and Hindsight.

It is now 30 years since the first report of a potent zinc-dependent histone deacetylase (HDAC) inhibitor appeared. Since then, five HDAC inhibitors have received regulatory approval for cancer chemotherapy while many others are in clinical development for oncology as well as other therapeutic indications. This Perspective reviews the biological and medicinal chemistry advances over the past 3 decades with an emphasis on the design of selective inhibitors that discriminate between the 11 human HDAC isoforms.