DXP Synthase‐Catalyzed CN Bond Formation: Nitroso Substrate Specificity Studies Guide Selective Inhibitor Design

Abstract

1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes the first step in the non-mammalian isoprenoid biosynthetic pathway to form DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate-dependent manner. Its unique structure and mechanism distinguish DXP synthase from its homologs, suggesting it should be pursued as an anti-infective drug target. However, few reports describe development of selective inhibitors of this enzyme. Here, we reveal a function of DXP synthase that catalyzes C-N bond formation and exploit aromatic nitroso substrates as active site probes. Substrate specificity studies reveal high affinity of DXP synthase for aromatic nitroso substrates compared to the related ThDP-dependent enzyme Pyruvate Dehydrogenase (PDH). Results from inhibition and mutagenesis studies indicate nitroso substrates bind to E. coli DXP synthase in a manner distinct from d-GAP. Our results suggest that incorporation of aryl acceptor substrate mimics into unnatural bisubstrate analogs will impart selectivity to DXP synthase inhibitors. As proof of concept, we show selective inhibition of DXP synthase by benzylacetylphosphonate (BnAP).

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

“…15,18 We hypothesized that increasing the alkyl-chain length on the AP scaffold would also lead to increased selectivity of inhibition against DXP synthase, as this enzyme possesses a larger active site compared to PDH and other related ThDP-dependent enzymes. 18 Thus, the inhibitory activities of 1 − 7 against PDH were compared. Compounds 1 − 7 are reversible competitive inhibitors with respect to pyruvate (Figures S4 and S6) and exhibit significantly weaker inhibitory activity against mammalian PDH compared to DXP synthase (Table 1).…”

Hydroxybenzaldoximes Are D‐GAP‐Competitive Inhibitors of E. coli 1‐Deoxy‐D‐Xylulose‐5‐Phosphate Synthase

“…This observation suggests that fluorine exerts electronic effects on the 4- and 5- hydroxyl groups that are comparable to the effect of incorporating a 2-hydroxy substituent. Given the large volume of the DXP synthase active site and propensity for sterically demanding alternative acceptor substrates to adopt distinct binding modes compared to d -GAP, 15 it is reasonable to suggest some oximes tested here may adopt binding modes which do not preclude binding of d -GAP.…”

“…15,21 Prior to the screening of the library at large, the diglyoxylate symmetrical dioximes 2a–d , present in all wells, were prepared individually by reacting 2 molar equivalents of glyoxylate with 1 molar equivalent of each dialkoxyammonium linker; these were tested for inhibition against DXP synthase and confirmed to be inactive up to 1 m m (data not shown). Oxime mixtures displaying > 50% inhibition at a total oxime concentration of 100 µ m were evaluated further.…”

“…Contrary to our expectations, unnatural bisubstrate analog inhibitors were not identified; rather, trihydroxybenzaldoximes emerged as new d -GAP competitive inhibitors with the 2,4,5-trihydroxybenzaldoxime scaffold exhibiting the most potent inhibitory activity. To date, the few inhibitors of DXP synthase described 15,16,18–20,34,35 exhibit micromolar inhibitory activities with K i values ranging from ~4 µ m (acetylphosphonates) to 75 µ m (ketoclomazone). Here, we have shown that oxime-based inhibitors display inhibitory potencies with K i values as low as 1 µ m , placing them amongst the most potent inhibitors of DXP synthase described to date (Table 1).…”

De novo fragment-based design of inhibitors of DXS guided by spin-diffusion-based NMR spectroscopy

“…S1, † 20% identity overall, 47% identity in the TDP-binding pocket between human TK and M. tuberculosis DXS). On the other hand, DXS has distinctive features compared to other TDP-dependent enzymes suggesting that selective inhibition of DXS over other TDP-dependent enzymes could be possible: the peculiar domain arrangement of Deinococcus radiodurans DXS (RCSB Protein Data Bank (PDB) code: 2O1X) where the TDP-binding site is located within the same monomer 24 and not at the dimer interface like in its mammalian homologues, 26 the particularly large active site 27 and its unique catalytic mechanism, which requires the formation of a ternary complex with pyruvate and GAP 28 (in contrast, all the other TDP-dependent enzymes follow classical ping-pong kinetics in which the binding of acceptor substrate is preceded by the activation of pyruvate and release of CO 2 ). 29 Therefore, ideally, a DXS-inhibitor should be designed so as to target both the TDPbinding pocket to enhance the affinity and the substrate-binding pocket to afford selectivity.…”