Disruption of an Active Site Network Leads to Activation of C2α-Lactylthiamin Diphosphate on the Antibacterial Target 1-Deoxy-d-xylulose-5-phosphate Synthase.

The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate (d-GAP). DXP is an essential bacteria-specific metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides with LThDP decarboxylation. This gated mechanism distinguishes DXPS in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate, while other pyruvate decarboxylases readily activate LThDP for decarboxylation, is a long-standing question in the field. We propose that an active site network functions to prevent LThDP activation on DXPS until the cosubstrate binds. Binding of d-GAP coincides with a conformational shift and disrupts the network causing changes in the active site that promote LThDP activation. Here, we show that the substitution of putative network residues, as well as nearby residues believed to contribute to network charge distribution, predictably affects LThDP reactivity. Substitutions predicted to disrupt the network have the effect to activate LThDP for decarboxylation, resulting in CO2 and acetate production. In contrast, a substitution predicted to strengthen the network fails to activate LThDP and has the effect to shift DXPS toward the closed state. Network-disrupting substitutions near the carboxylate of LThDP also have a pronounced effect to shift DXPS to an open state. These results offer initial insights to explain the long-lived LThDP intermediate and its activation through disruption of an active site network, which is unique to DXPS. These findings have important implications for DXPS function in bacteria and its development as an antibacterial target.

Aldehyde-based activation of C2α-lactylthiamin diphosphate decarboxylation on bacterial 1-deoxy-d-xylulose 5-phosphate synthase.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate (donor substrate) and d-glyceraldehyde 3-phosphate (d-GAP, acceptor substrate) in bacterial central metabolism. DXPS uses a ligand-gated mechanism in which binding of a small molecule "trigger" activates the first enzyme-bound intermediate, C2α-lactylThDP (LThDP), to form the reactive carbanion via LThDP decarboxylation. d-GAP is the natural acceptor substrate for DXPS and also serves a role as a trigger to induce LThDP decarboxylation in the gated step. Additionally, we have shown that O2 and d-glyceraldehyde (d-GA) can induce LThDP decarboxylation. We hypothesize this ligand-gated mechanism poises DXPS to sense and respond to cellular cues in metabolic remodeling during bacterial adaptation. Here we sought to characterize features of small molecule inducers of LThDP decarboxylation. Using a combination of CD, NMR and biochemical methods, we demonstrate that the α-hydroxy aldehyde moiety of d-GAP is sufficient to induce LThDP decarboxylation en route to DXP formation. A variety of aliphatic aldehydes also induce LThDP decarboxylation. The study highlights the capacity of DXPS to respond to different molecular cues, lending support to potential multifunctionality of DXPS and its metabolic regulation by this mechanism.

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction.

The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2-C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O2, H+, and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C-C bond-forming enzymes.

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism.

The thiamin diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d-glyceraldehyde 3-phosphate (d-GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d-GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor-acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.

X-ray crystallography-based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) uses thiamine diphosphate (ThDP) to convert pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) into 1-deoxy-d-xylulose 5-phosphate (DXP), an essential bacterial metabolite. DXP is not utilized by humans; hence, DXPS has been an attractive antibacterial target. Here, we investigate DXPS from Deinococcus radiodurans (DrDXPS), showing that it has similar kinetic parameters Kmd-GAP and Kmpyruvate (54 ± 3 and 11 ± 1 µm, respectively) and comparable catalytic activity (kcat = 45 ± 2 min-1) with previously studied bacterial DXPS enzymes and employing it to obtain missing structural data on this enzyme family. In particular, we have determined crystallographic snapshots of DrDXPS in two states along the reaction coordinate: a structure of DrDXPS bound to C2α-phosphonolactylThDP (PLThDP), mimicking the native pre-decarboxylation intermediate C2α-lactylThDP (LThDP), and a native post-decarboxylation state with a bound enamine intermediate. The 1.94-Å-resolution structure of PLThDP-bound DrDXPS delineates how two active-site histidine residues stabilize the LThDP intermediate. Meanwhile, the 2.40-Å-resolution structure of an enamine intermediate-bound DrDXPS reveals how a previously unknown 17-Å conformational change removes one of the two histidine residues from the active site, likely triggering LThDP decarboxylation to form the enamine intermediate. These results provide insight into how the bi-substrate enzyme DXPS limits side reactions by arresting the reaction on the less reactive LThDP intermediate when its cosubstrate is absent. They also offer a molecular basis for previous low-resolution experimental observations that correlate decarboxylation of LThDP with protein conformational changes.

Active Site Histidines Link Conformational Dynamics with Catalysis on Anti-Infective Target 1-Deoxy-d-xylulose 5-Phosphate Synthase.

The product of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase, DXP, feeds into the bacterial biosynthesis of isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate. DXP is essential for human pathogens but not utilized by humans; thus, DXP synthase is an attractive anti-infective target. The unique ThDP-dependent mechanism and structure of DXP synthase offer ideal opportunities for selective targeting. Upon reaction with pyruvate, DXP synthase uniquely stabilizes the predecarboxylation intermediate, C2α-lactylThDP (LThDP), in a closed conformation. Subsequent binding of d-glyceraldehyde 3-phosphate induces an open conformation that is proposed to destabilize LThDP, triggering decarboxylation. Evidence for the closed and open conformations has been revealed by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography, which indicate that H49 and H299 are involved in conformational dynamics and movement of the fork and spoon motifs away from the active site is important for the closed-to-open transition. Interestingly, H49 and H299 are critical for DXP formation and interact with the predecarboxylation intermediate in the closed conformation. H299 is removed from the active site in the open conformation of the postdecarboxylation state. In this study, we show that substitution at H49 and H299 negatively impacts LThDP formation by shifting the conformational equilibrium of DXP synthase toward an open conformation. We also present a method for monitoring the dynamics of the spoon motif that uncovered a previously undetected role for H49 in coordinating the closed conformation. Overall, our results suggest that H49 and H299 are critical for the closed, predecarboxylation state providing the first direct link between catalysis and conformational dynamics.

Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria.

The underexploited antibacterial target 1-deoxy-d-xyluose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP). DXP is an essential intermediate in the biosynthesis of ThDP, pyridoxal phosphate, and isoprenoids in many pathogenic bacteria. DXP synthase catalyzes a distinct mechanism in ThDP decarboxylative enzymology in which the first enzyme-bound pre-decarboxylation intermediate, C2α-lactyl-ThDP (LThDP), is stabilized by DXP synthase in the absence of d-GAP, and d-GAP then induces efficient LThDP decarboxylation. Despite the observed LThDP accumulation and lack of evidence for C2α-carbanion formation in the absence of d-GAP, CO2 is released at appreciable levels under these conditions. Here, seeking to resolve these conflicting observations, we show that DXP synthase catalyzes the oxidative decarboxylation of pyruvate under conditions in which LThDP accumulates. O2-dependent LThDP decarboxylation led to one-electron transfer from the C2α-carbanion/enamine to O2, with intermediate ThDP-enamine radical formation, followed by peracetic acid formation en route to acetate. Thus, LThDP formation and decarboxylation and DXP formation were studied under anaerobic conditions. Our results support a model in which O2-dependent LThDP decarboxylation and peracetic acid formation occur in the absence of d-GAP, decreasing the levels of pyruvate and O2 in solution. The relative pyruvate and O2 concentrations then dictate the extent of LThDP accumulation, and its buildup can be observed when [pyruvate] > [O2]. The finding that O2 acts as a structurally distinct trigger of LThDP decarboxylation supports the hypothesis that a mechanism involving small molecule-dependent LThDP decarboxylation equips DXP synthase for diverse, yet uncharacterized cellular functions.

Toward Understanding the Chemistry and Biology of 1-Deoxy-d-xylulose 5-Phosphate (DXP) Synthase: A Unique Antimicrobial Target at the Heart of Bacterial Metabolism.

Antibiotics are the cornerstone of modern healthcare. The 20th century discovery of sulfonamides and ß-lactam antibiotics altered human society immensely. Simple bacterial infections were no longer a leading cause of morbidity and mortality, and antibiotic prophylaxis greatly reduced the risk of infection from surgery. The current healthcare system requires effective antibiotics to function. However, antibiotic-resistant infections are becoming increasingly prevalent, threatening the emergence of a postantibiotic era. To prevent this public health crisis, antibiotics with novel modes of action are needed. Currently available antibiotics target just a few cellular processes to exert their activity: DNA, RNA, protein, and cell wall biosynthesis. Bacterial central metabolism is underexploited, offering a wealth of potential new targets that can be pursued toward expanding the armamentarium against microbial infections. Discovered in 1997 as the first enzyme in the methylerythritol phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylative condensation of pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to form DXP. This five-carbon metabolite feeds into three separate essential pathways for bacterial central metabolism: ThDP synthesis, pyridoxal phosphate (PLP) synthesis, and the MEP pathway for isoprenoid synthesis. While it has long been identified as a target for the development of antimicrobial agents, limited progress has been made toward developing selective inhibitors of the enzyme. This Account highlights advances from our lab over the past decade to understand this important and unique enzyme. Unlike all other known ThDP-dependent enzymes, DXP synthase uses a random-sequential mechanism that requires the formation of a ternary complex prior to decarboxylation of the lactyl-ThDP intermediate. Its large active site accommodates a variety of acceptor substrates, lending itself to a number of alternative activities, such as the production of α-hydroxy ketones, hydroxamates, amides, acetolactate, and peracetate. Knowledge gained from mechanistic and substrate-specificity studies has guided the development of selective inhibitors with antibacterial activity and provides a biochemical foundation toward understanding DXP synthase function in bacterial cells. Although it is a promising drug target, the centrality of DXP synthase in bacterial metabolism imparts specific challenges to assessing antibacterial activity of DXP synthase inhibitors, and the susceptibility of most bacteria to current DXP synthase inhibitors is remarkably culture-medium-dependent. Despite these challenges, the study of DXP synthase is poised to reveal the role of DXP synthase in bacterial metabolic adaptability during infection, ultimately providing a more complete picture of how inhibiting this crucial enzyme can be used to develop novel antibiotics.

Targeting the Unique Mechanism of Bacterial 1-Deoxy-d-xylulose-5-phosphate Synthase.

The bacterial metabolite 1-deoxy-d-xyulose 5-phosphate (DXP) is essential in bacterial central metabolism feeding into isoprenoid, thiamin diphosphate (ThDP), and pyridoxal phosphate de novo biosynthesis. Halting its production through the inhibition of DXP synthase is an attractive strategy for the development of novel antibiotics. Recent work has revealed that DXP synthase utilizes a unique random sequential mechanism that requires formation of a ternary complex among pyruvate-derived C2α-lactylthiamin diphosphate (LThDP), d-glyceraldehyde 3-phosphate (d-GAP), and enzyme, setting it apart from all other known ThDP-dependent enzymes. Herein, we describe the development of bisubstrate inhibitors bearing an acetylphosphonate (AP) pyruvate mimic and a distal negative charge mimicking the phosphoryl group of d-GAP, designed to target the unique form of DXP synthase that binds LThDP and d-GAP in a ternary complex. A d-phenylalanine-derived triazole acetylphosphonate (d-PheTrAP) emerged as the most potent inhibitor in this series, displaying slow, tight-binding inhibition with a Ki* of 90 ± 10 nM, forward ( k1) and reverse ( k2) isomerization rates of 1.1 and 0.14 min-1, respectively, and exquisite selectivity (>15000-fold) for DXP synthase over mammalian pyruvate dehydrogenase. d-PheTrAP is the most potent, selective DXP synthase inhibitor described to date and represents the first inhibitor class designed specifically to exploit the unique E-LThDP-GAP ternary complex in ThDP enzymology.

A novel lipoate attachment enzyme is shared by Plasmodium and Chlamydia species.

Lipoate is an essential cofactor for enzymes that are important for central metabolism and other processes. In malaria parasites, scavenged lipoate from the human host is required for survival. The Plasmodium falciparum mitochondrion contains two enzymes (PfLipL1 and PfLipL2) that are responsible for activating mitochondrial proteins through the covalent attachment of lipoate (lipoylation). Lipoylation occurs via a novel redox-gated mechanism that remains poorly understood. We show that PfLipL1 functions as a redox switch that determines which downstream proteins will be activated. Based on the lipoate redox state, PfLipL1 either functions as a canonical lipoate ligase or as a lipoate activating enzyme which works in conjunction with PfLipL2. We demonstrate that PfLipL2 is a lipoyltransferase and is a member of a novel clade of lipoate attachment enzymes. We show that a LipL2 enzyme from Chlamydia trachomatis has similar activity, demonstrating conservation between intracellular pathogens from different phylogenetic kingdoms and supporting the hypothesis that an early ancestor of malaria parasites once contained a chlamydial endosymbiont. Redox-dependent lipoylation may regulate processes such as central metabolism and oxidative defense pathways.

Synthesis and Evaluation of Fluoroalkyl Phosphonyl Analogues of 2- C-Methylerythritol Phosphate as Substrates and Inhibitors of IspD from Human Pathogens.

Targeting essential bacterial processes beyond cell wall, protein, nucleotide, and folate syntheses holds promise to reveal new antimicrobial agents and expand the potential drugs available for combination therapies. The synthesis of isoprenoid precursors, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), is vital for all organisms; however, humans use the mevalonate pathway for production of IDP/DMADP while many pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis, use the orthogonal methylerythritol phosphate (MEP) pathway. Toward developing novel antimicrobial agents, we have designed and synthesized a series of phosphonyl analogues of MEP and evaluated their abilities to interact with IspD, both as inhibitors of the natural reaction and as antimetabolite alternative substrates that could be processed enzymatically to form stable phosphonyl analogues as potential inhibitors of downstream MEP pathway intermediates. In this compound series, the S-monofluoro MEP analogue displays the most potent inhibitory activity against Escherichia coli IspD and is the best substrate for both the E. coli and P. falciparum IspD orthologues with a Km approaching that of the natural substrate for the E. coli enzyme. This work represents a first step toward the development of phosphonyl MEP antimetabolites to modulate early isoprenoid biosynthesis in human pathogens.

Pharmacological activation of myosin II paralogs to correct cell mechanics defects.

Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.

Redox-dependent lipoylation of mitochondrial proteins in Plasmodium falciparum.

Lipoate scavenging from the human host is essential for malaria parasite survival. Scavenged lipoate is covalently attached to three parasite proteins: the H-protein and the E2 subunits of branched chain amino acid dehydrogenase (BCDH) and α-ketoglutarate dehydrogenase (KDH). We show mitochondrial localization for the E2 subunits of BCDH and KDH, similar to previously localized H-protein, demonstrating that all three lipoylated proteins reside in the parasite mitochondrion. The lipoate ligase 1, LipL1, has been shown to reside in the mitochondrion and it catalyses the lipoylation of the H-protein; however, we show that LipL1 alone cannot lipoylate BCDH or KDH. A second mitochondrial protein with homology to lipoate ligases, LipL2, does not show ligase activity and is not capable of lipoylating any of the mitochondrial substrates. Instead, BCDH and KDH are lipoylated through a novel mechanism requiring both LipL1 and LipL2. This mechanism is sensitive to redox conditions where BCDH and KDH are exclusively lipoylated under strong reducing conditions in contrast to the H-protein which is preferentially lipoylated under less reducing conditions. Thus, malaria parasites contain two different routes of mitochondrial lipoylation, an arrangement that has not been described for any other organism.

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

1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low-micromolar inhibitors, competitive against D-glyceraldehyde 3-phosphate (D-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D-GAP-competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis.

Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog.

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)-based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure-activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

Investigating inhibitors of 1-deoxy-d-xylulose 5-phosphate synthase in a mouse model of UTI.

The rising rate of antimicrobial resistance continues to threaten global public health. Further hastening antimicrobial resistance is the lack of new antibiotics against new targets. The bacterial enzyme, 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), is thought to play important roles in central metabolism, including processes required for pathogen adaptation to fluctuating host environments. Thus, impairing DXPS function represents a possible new antibacterial strategy. We previously investigated a DXPS-dependent metabolic adaptation as a potential target in uropathogenic Escherichia coli (UPEC) associated with urinary tract infection (UTI), using the DXPS-selective inhibitor butyl acetylphosphonate (BAP). However, investigations of DXPS inhibitors in vivo have not been conducted. The goal of the present study is to advance DXPS inhibitors as in vivo probes and assess the potential of inhibiting DXPS as a strategy to prevent UTI in vivo. We show that BAP was well-tolerated at high doses in mice and displayed a favorable pharmacokinetic profile for studies in a mouse model of UTI. Further, an alkyl acetylphosphonate prodrug (homopropargyl acetylphosphonate, pro-hpAP) was significantly more potent against UPEC in urine culture and exhibited good exposure in the urinary tract after systemic dosing. Prophylactic treatment with either BAP or pro-hpAP led to a partial protective effect against UTI, with the prodrug displaying improved efficacy compared to BAP. Overall, our results highlight the potential for DXPS inhibitors as in vivo probes and establish preliminary evidence that inhibiting DXPS impairs UPEC colonization in a mouse model of UTI.IMPORTANCENew antibiotics against new targets are needed to prevent an antimicrobial resistance crisis. Unfortunately, antibiotic discovery has slowed, and many newly FDA-approved antibiotics do not inhibit new targets. Alkyl acetylphosphonates (alkyl APs), which inhibit the enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), represent a new possible class of compounds as there are no FDA-approved DXPS inhibitors. To our knowledge, this is the first study demonstrating the in vivo safety, pharmacokinetics, and efficacy of alkyl APs in a urinary tract infection mouse model.

DXP synthase-catalyzed C-N bond formation: nitroso substrate specificity studies guide selective inhibitor design.

1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the first step in the nonmammalian 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 homologues and suggest that it should be pursued as an anti-infective drug target. However, few reports describe any development of selective inhibitors of this enzyme. Here, we reveal that DXP synthase catalyzes C-N bond formation and exploit aromatic nitroso substrates as active site probes. Substrate specificity studies reveal a 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 that nitroso substrates bind to E. coli DXP synthase in a manner distinct from that of D-GAP. Our results suggest that the incorporation of aryl acceptor substrate mimics into unnatural bisubstrate analogues will impart selectivity to DXP synthase inhibitors. As a proof of concept, we show selective inhibition of DXP synthase by benzylacetylphosphonate (BnAP).

DXP Synthase Function in a Bacterial Metabolic Adaptation and Implications for Antibacterial Strategies.

Pathogenic bacteria possess a remarkable ability to adapt to fluctuating host environments and cause infection. Disturbing bacterial central metabolism through inhibition of 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) has the potential to hinder bacterial adaptation, representing a new antibacterial strategy. DXPS functions at a critical metabolic branchpoint to produce the metabolite DXP, a precursor to pyridoxal-5-phosphate (PLP), thiamin diphosphate (ThDP) and isoprenoids presumed essential for metabolic adaptation in nutrient-limited host environments. However, specific roles of DXPS in bacterial adaptations that rely on vitamins or isoprenoids have not been studied. Here we investigate DXPS function in an adaptation of uropathogenic E. coli (UPEC) to d-serine (d-Ser), a bacteriostatic host metabolite that is present at high concentrations in the urinary tract. UPEC adapt to d-Ser by producing a PLP-dependent deaminase, DsdA, that converts d-Ser to pyruvate, pointing to a role for DXPS-dependent PLP synthesis in this adaptation. Using a DXPS-selective probe, butyl acetylphosphonate (BAP), and leveraging the toxic effects of d-Ser, we reveal a link between DXPS activity and d-Ser catabolism. We find that UPEC are sensitized to d-Ser and produce sustained higher levels of DsdA to catabolize d-Ser in the presence of BAP. In addition, BAP activity in the presence of d-Ser is suppressed by ß-alanine, the product of aspartate decarboxylase PanD targeted by d-Ser. This BAP-dependent sensitivity to d-Ser marks a metabolic vulnerability that can be exploited to design combination therapies. As a starting point, we show that combining inhibitors of DXPS and CoA biosynthesis displays synergy against UPEC grown in urine where there is increased dependence on the TCA cycle and gluconeogenesis from amino acids. Thus, this study provides the first evidence for a DXPS-dependent metabolic adaptation in a bacterial pathogen and demonstrates how this might be leveraged for development of antibacterial strategies against clinically relevant pathogens.

Preclinical Evaluation of Long-Acting Emtricitabine Semi-Solid Prodrug Nanoparticle Formulations.

Long-acting injectable (LAI) formulations promise to deliver patient benefits by overcoming issues associated with non-adherence. A preclinical assessment of semi-solid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) is reported here. Pharmacokinetics over 28 days were assessed in Wistar rats, New Zealand white rabbits, and Balb/C mice following intramuscular injection. Two lead formulations were assessed for the prevention of an HIV infection in NSG-cmah-/- humanised mice to ensure antiviral activities were as anticipated according to the pharmacokinetics. Cmax was reached by 12, 48, and 24 h in rats, rabbits, and mice, respectively. Plasma concentrations were below the limit of detection (2 ng/mL) by 21 days in rats and rabbits, and 28 days in mice. Mice treated with SSPN formulations demonstrated undetectable viral loads (700 copies/mL detection limit), and HIV RNA remained undetectable 28 days post-infection in plasma, spleen, lung, and liver. The in vivo data presented here demonstrate that the combined prodrug/SSPN approach can provide a dramatically extended pharmacokinetic half-life across multiple preclinical species. Species differences in renal clearance of FTC mean that longer exposures are likely to be achievable in humans than in preclinical models.

Observation of thiamin-bound intermediates and microscopic rate constants for their interconversion on 1-deoxy-D-xylulose 5-phosphate synthase: 600-fold rate acceleration of pyruvate decarboxylation by D-glyceraldehyde-3-phosphate.

The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design.