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Scheme 3. Synthesis of C2-acylated furan-based esters. Reagents and conditions: i) carboxylic acid, DCC, DMAP, DMF, RT; ii) RCOCl, AlCl 3 , DCM, RT; iii) acetic anhydride, pyridine, 40 o C; iv) K 2 CO 3 , MeOH, RT.
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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-lim...
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Context 1
... benzene ring forms a pi-cation interaction with the Mg 2+ and the nitrogen atom of the pyridine ring hydrogen bonds to the amide NH of an arginine residue, explaining why the m-pyridin-3-yl group of 8w binds better than the m-phenyl group of 8v. The C2-butanoyl group makes only hydrophobic contacts in the C2-pocket and the aminopyrimidine ring forms all the same hydrogen bonds and pi-pi interaction with surrounding residues that TPP does (Figure 2b, see also Figure S3). ...Context 2
... 37 is a slightly stronger inhibitor, its aqueous solubility is very low, so the more water-soluble 33 was selected and tested at 100 μM on four fast-growing human cell lines. Amide 33 showed no significant effect on cell-viability of any of the cell lines ( Figure S5) but a marked cytostatic effect on two of the cell lines, growth-inhibition on a third and no effect on the fourth (Figure 3). The cytostatic effect might be due to inhibition of PDHc, in which case greater suppression on cell growth in HEK and MDA-MB-231 cell lines (relative to the other two cell lines) may be because they rely more on PDHcmediated oxygen-dependent metabolic pathways for providing energy to sustain their growth. ...Citations
... The charged ring-opened series would suffer a greater desolvation penalty than the cyclic series upon leaving the aqueous environment and binding in this hydrophobic region. 31,[43][44][45][46] If the highly polar moiety of the ring-opened series were able to interact with the Mg 2+ ion in the diphosphate pocket, they would be expected to bind better than the cyclic series, so we suggest that the side chain is not long enough to allow any effective interaction with the Mg 2+ ion. ...
... Although one possible explanation would be that 2a and 3a are weak inhibitors of human PDH E1 despite their potent inhibition on porcine PDH E1, it is generally accepted that the latter is a good model for the former because the sequence of the two enzymes are >95% identical and the residues that differ are located away from the active site. 44 Another possible explanation would be due to hydrolysis of 2a into 3a. 25 Extracellular hydrolysis would lead to reduced cell entry of inhibitors as 3a is almost membrane-impermeable (fraction absorbed = 6% in Parallel Artificial Membrane Permeability Assay, PAMPA, 50 as shown in Table S2). ...
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.
... In our recent paper, several matched molecular pairs revealed that changing the central ring from triazole to furan consistently improved binding affinities. 58 This prompted us to look into the role of the central ring of thiamine/ThDP analogues in inhibiting ThDP-dependent enzymes. ...
... Thiamine and its analogues are weak binders, as the tight binding of ThDP to the enzymes is largely through the ionic interactions with the Mg 2+ cation in the diphosphate pocket ( Figure 1a). Thiamine analogues 1a-f were tosylated and then di-phosphorylated to yield ThDP analogues 2, coupled with p-chlorobenzoic acid to yield esters 3a-f (as we have recently identified 3b as a potent and selective PDH E1 inhibitor 58 ), and monophosphorylated to yield ThMP analogues 4 (the first reported ThMP analogues). The syntheses of pyrro-ThDP (2d) and pyrro-ThMP (4d) were unsuccessful, due (we believe) to the electron-rich pyrrole ring making these compounds (and the intermediates) unstable. ...
... The 22 derivatives were tested for inhibiton of four ThDP-dependent enzymes, namely PDH E1, PDC, PO and OGDH E1 (Table 1 and Figures S1-4). Analogues 1-4 have been validated as ThDP-competitive inhibitors because their apparent inhibition decreased under increasing levels of ThDP (see SI and previous reports 26,27,29,30,58 ); therefore, their Ki values were calculated based on the measured KM of ThDP for each individual enzyme. ...
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 positive 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) among the reported aromatic rings, which is the best? 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 improve selectivity.
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.
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.
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.
