Caren L. Freel Meyers

Background/Objectives: Long-acting therapeutics are being investigated for their potential to enhance medication adherence and improve overall treatment outcomes. Due to the prolonged residence time at the administration site, such as subcutaneous insertion, long-acting preparations may modulate the responses of local immune cells linked to material characteristics. Long-acting Polymer of Prodrug (POP) of Nucleoside Reverse Transcriptase Inhibitor (NRTI) Emtricitabine (FTC) has been developed for subcutaneous administration. Methods: The present study focused on the acute immunocompatibility of an FTC-based POP and its interactions with various cells and components of the immune system following acute exposure using DMSO-dispersed polymer linear poly(FTC). A tiered assessment was carried out, including assessment of pyrogens (MAT), cytotoxicity (MTT/LDH), Leukocyte proliferation (MTT), complement proteins (iC3b, Factor H, sC5b-9, Bb and C4d EIAs) and cytokine secretion in whole blood (Luminex panel). Results: Linear poly(FTC) had no detectable pyrogenic activity, no overt cytotoxicity within the THP-1 cell line, no consistent leukocyte proliferative activation and no broad whole-blood cytokine induction. Complement analysis, however, indicated a concentration-associated higher iC3b signal by linear poly(FTC) and a higher Bb signal by linear poly(FTC) at 80 µg/mL when compared to vehicle (0.4% DMSO). Conclusions: Further investigations are warranted to assess the long-term implications of these implants, given their extended residence times. Therefore, the adaptation of existing safety assessments should be prioritized to facilitate the safe translation of these POPs into clinical studies.

The thiamine diphosphate (ThDP)-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to the essential branchpoint metabolite, DXP. DXPS exhibits a ligand-gated mechanism where the predecarboxylation intermediate derived from pyruvate persists until d-GAP triggers decarboxylation. Together with its substrate/catalytic promiscuity and other features of multifunctionality, ligand gating on DXPS may poise this enzyme to catalyze alternate activities that aid in bacterial adaptation. We previously discovered alternative donors for Escherichia coli DXPS, with donor processing and acceptor specificity influenced by the donor. Here, we demonstrate species differences in DXPS substrate usage and processing which could signify that DXPS homologues support bacterial adaptation in distinct ways. We showed that hydroxypyruvate (HPA) is a donor for Pseudomonas aeruginosa DXPS (PaDXPS), similar to EcDXPS, yet HPA processing and donor-acceptor specificity differs on these homologues; HPA reacts with ThDP on PaDXPS to form a longer-lived intermediate in the absence of added trigger, relative to EcDXPS. Notably, PaDXPS preferentially catalyzes xylulose 5-phosphate formation from HPA and d-GAP, in contrast to EcDXPS, suggesting a potential connection between DXPS activity and pentose phosphate pathway metabolism in P. aeruginosa. Finally, both EcDXPS and PaDXPS were found to generate a 5-carbon β-ketoacid via a one-substrate reaction of HPA, distinguishing DXPS from transketolase (TK) which produces erythrulose under similar conditions. This study illustrates species differences in DXPS donor usage and processing, and donor-acceptor specificity that supports distinct multifunctionality across bacterial pathogens and the plausibility of species-specific targeting.

The essential bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is absent in humans, making the enzyme an attractive antimicrobial target. Its product DXP sits at a metabolic branchpoint and is required for biosynthesis of pyridoxal phosphate (PLP), thiamin diphosphate (ThDP), and isoprenoids. DXP is formed via decarboxylation of pyruvate and subsequent carboligation with d-glyceraldehyde-3-phosphate (d-GAP) in a ThDP-dependent manner. In the current mechanistic model, DXPS follows a ligand-gated mechanism. Pyruvate reacts with ThDP to form C2α-lactylThDP (LThDP), which coincides with a shift to a closed conformation. The flexible "spoon" and "fork" motifs become ordered and the catalytic residue H299 is placed within the active site, supporting LThDP formation and persistence in the closed conformation of the E-LThDP complex until binding of d-GAP. We aim to understand the molecular basis for stabilization of the E-LThDP complex in its closed conformation. We propose that the conserved aromatic residues Y288, F298, and F304 in theE. coli DXPS spoon and fork motifs form a cluster upon transition from the open to closed form, positioning H299 within the active site where it has roles in LThDP formation and persistence. We conducted mutagenesis studies to elucidate the roles of Y288, F298, and F304 in conformational cycling and catalysis. Variants (1) adopted open conformations, (2) displayed significantly reduced k_cat, (3) promoted LThDP decarboxylation, and (4) exhibited decreased affinity for the postdecarboxylation intermediate. Our results support a model in which conserved aromatic residues within the mobile, sequence-diverse spoon/fork motifs promote the closed conformation, supporting catalysis and LThDP persistence.

The enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first and rate-limiting step of the methylerythritol 4-phosphate (MEP) pathway, representing a promising target for novel anti-infective agents. Given its essential role in the survival of Gram-negative pathogenic bacteria and its absence in humans, drug-discovery efforts to advance our understanding of this enzyme are urgently needed. Here, we unraveled a novel druggable allosteric pocket in DXPS, unexpectedly revealed through co-crystallization of tool compound 14 with Pseudomonas aeruginosa DXPS. This inhibitor, identified via virtual screening and subsequent synthetic optimization, binds within an allosteric site distinct from the active site, engaging the protein through halogen bonding interactions. Compound 14 exhibits comparable IC₅₀ values against both P. aeruginosa and Klebsiella pneumoniae DXPS, highlighting its potential as a broad-spectrum DXPS inhibitor. This first co-crystal structure of a non-substrate analog inhibitor with a pathogenic DXPS establishes 14 as a valuable tool compound and provides a novel structural template for future antibiotic development.

The essential bacterial enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) is absent in humans, making the enzyme an attractive antimicrobial target. Its product DXP sits at a metabolic branchpoint between the biosynthesis of pyridoxal phosphate (PLP), thiamin diphosphate (ThDP), and isoprenoids. DXP is formed via decarboxylation of pyruvate and subsequent carboligation with D-glyceraldehyde-3-phosphate (D-GAP) in a ThDP-dependent manner. In the current mechanistic model, DXPS follows a ligand-gated mechanism. Pyruvate reacts with ThDP to form C2⍺-lactylThDP (LThDP) which coincides with a shift to a closed conformation. The flexible “spoon” and “fork” motifs become ordered, situating the catalytic residue H299 within the active site which supports LThDP persistence and the closed conformation of the E-LThDP complex until binding of D-GAP. Our goal is to understand the molecular basis for stabilization of the E-LThDP complex in its closed conformation in the absence of D-GAP. We propose the conserved aromatic residues Y288, F298, and F304 in the E. coli DXPS spoon and fork motifs form a cluster upon transition from the open to closed form to position H299 within the active site, necessary for LThDP persistence. Here, we conducted mutagenesis studies to elucidate the roles of Y288, F298, and F304 in conformational cycling and catalysis. On each variant, the conformational equilibrium favored an open state, hindered intermediate formation and persistence, and promoted intermediate release from the active site. Our results support a model in which conserved aromatic residues within the mobile, sequence-diverse spoon and fork motifs promote the closed conformation and support catalysis.

Corrigendum on: Coco LB and Freel Meyers CL (2024) An activity-based probe for antimicrobial target DXP synthase, a thiamin diphosphate-dependent enzyme. Front. Chem. Biol 3:1389620. doi: 10.3389/fchbi.2024.1389620In the published article, there was an error. The concentrations of click reaction components were inadvertently reported as 10-fold higher than actually used for labeling experiments using purified recombinant DXPS. A correction has been made to "General protocol for DXPS labeling by 1", Section 4.4. This sentence previously stated: "Solutions were cooled to ambient temperature, and a 7.5x CuAAC reaction stock (4 uL, prepared immediately prior to addition) was added to achieve final concentrations of 1 mM tris ((1-benzyl-4-triazolyl)methyl)amine (TBTA), 10 mM CuSO4, and 10 mM tris(2carboxyethyl)phosphine (TCEP)."The corrected sentence appears below: "Solutions were cooled to ambient temperature, and a 7.5x CuAAC reaction stock (4 uL, prepared immediately prior to addition) was added to achieve final concentrations of 0.1 mM tris ((1-benzyl-4-triazolyl)methyl)amine (TBTA), 1 mM CuSO4, and 1 mM tris(2carboxyethyl)phosphine (TCEP)."The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.In the published article, there was an error. The concentrations of click reaction components were inadvertently reported as 10-fold higher than actually used in experiments to label DXPS in bacterial lysate. A correction has been made to "Detection of DXPS in complex bacterial lysate" (Section 4.6), subsection "Labeling of DXPS by 1 in lysate" (sub-section 4.6.2), paragraph 1. This sentence previously stated:

Long-acting injectable (LAI) antiretroviral (ARV) approaches are needed to address poor adherence to oral drug regimens in the treatment and prevention of human immunodeficiency virus (HIV). We have previously reported semisolid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) that showed extended pharmacokinetic half-lives in vivo. Here, we report the design, synthesis, and evaluation of FTC octyl carbamate prodrugs masked with a variety of bioreversible protecting groups at the 5′-position. We show that 5′-modifications displaying a range of hydrolytic stabilities in biomatrices are compatible with the LAI formulation by the emulsion-templated freeze-drying (ETFD) method. The results demonstrate the feasibility of tuning prodrug activation kinetics while maintaining LAI compatibility, which is critical for the development of nucleoside reverse transcriptase inhibitor (NRTI)-containing LAI-ARV therapies.

Metabolic processes required for bacterial pathogens to adapt in the host are potential targets for the development of much-needed new antimicrobial agents. The bacterial central metabolic enzyme 1-deoxy- d -xylulose 5-phosphate synthase (DXPS) is required for the synthesis of essential isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate (PLP) and is believed to function in bacterial adaptations requiring these metabolites. DXPS inhibition impairs a PLP-dependent adaptation of Uropathogenic Escherichia coli (UPEC) to d -serine, a bacteriostatic host metabolite in urine that inhibits pantothenate production in Coenzyme A (CoA) biosynthesis. CoA is required for a functioning tricarboxylic acid (TCA cycle, which is critical for UPEC survival in the urinary tract. Accordingly, inhibition of DXPS also sensitizes UPEC to N -pentylpantothenamide (N5-Pan, 1 ), an inhibitor of CoA synthesis. However, 1 is enzymatically hydrolyzed by pantetheinases, presenting challenges for its use in in vivo studies. We sought to identify a pantothenamide inhibitor candidate for studies to explore the in vivo efficacy of inhibitor combinations targeting DXPS and CoA syntheses. Here, we describe studies that highlight a truncated analog of 1 , pantetheinase-resistant N5-α-Pan ( 6 ), as a promising candidate for in vivo studies. Analog 6 exhibited comparable antimicrobial activity with 1 against UPEC grown in nutrient-limited culture conditions, including urine, and displayed enhanced activity in the presence of a DXPS inhibitor. In contrast to 1 , analog 6 was stable in the presence of mouse plasma and liver enzymes, making it suitable for studies to investigate an inhibitor combination targeting DXPS and CoA synthesis in UPEC by the in vivo ascending UTI assay. IMPORTANCE: New approaches are needed to control the emergence of drug resistance in bacterial pathogens that cause life-threatening infections. Targeting DXPS-dependent synthesis of vitamins is a promising approach to prevent pathogen metabolic adaptation. Metabolic processes requiring DXPS-dependent synthesis of pyridoxal phosphate (PLP) or thiamin diphosphate (ThDP) can become particularly vulnerable in a specific pathogen and/or host environment, under conditions of DXPS inhibition. We previously observed that UPEC grown in urine is particularly sensitive to an inhibitor combination targeting DXPS and CoA synthesis; however, the CoA inhibitor is readily hydrolyzed by a host pantetheinase. This study is significant, as it identifies a pantothenamide inhibitor of CoA synthesis, N5-α-Pan ( 6 ), that is stable to mouse plasma and liver enzymes and whose activity is enhanced in the presence of a DXPS inhibitor. Thus, 6 is suitable for studies to explore how a pathogen can become sensitized in vivo under conditions of DXPS inhibition.

Pathogenic bacteria must swiftly adapt to dynamic infection environments in order to survive and colonize in the host. 1-Deoxy-d-xylulose-5-phosphate synthase (DXPS) is thought to play a critical role in bacterial adaptation during infection and is a promising drug target. DXPS utilizes a thiamine diphosphate (ThDP) cofactor to catalyze the decarboxylative condensation of pyruvate and d-glyceraldehyde-3-phosphate (d-GAP) to form DXP, a precursor to isoprenoids and B vitamins. DXPS follows a ligand-gated mechanism in which pyruvate reacts with ThDP to form a long-lived lactyl-ThDP (LThDP) adduct which is coordinated by an active-site network of residues. d-GAP binding ostensibly disrupts this network to activate LThDP for decarboxylation. Our lab previously reported trihydroxybenzaldoxime inhibitors which are competitive with respect to d-GAP, and uncompetitive with respect to pyruvate, suggesting they bind after E-LThDP complex formation. Here, we conducted mechanistic studies to determine if these compounds inhibit DXPS by preventing LThDP activation or if they act as inducers of LThDP activation. We discovered that the catechol moiety of the trihydroxybenzaldoxime scaffold undergoes oxidation under alkaline aerobic conditions, and inhibitory potency is reduced under oxygen restriction. Leveraging long-range ¹H-¹⁵N HSQC NMR and electrochemical measurements, we demonstrated that the oxidized form of the trihydroxybenzaldoxime induces LThDP decarboxylation and accepts electrons from the resulting carbanion, resulting in reduction to the catechol and formation of acetyl-ThDP which hydrolyzes to form acetate. Under aerobic conditions the catechol is reoxidized. Thus, these compounds act as redox cycling, substrate-wasting inhibitors of DXP formation. These findings uncover a novel activity and mechanism of DXPS inhibition which may have implications for DXPS-mediated redox activity in bacteria. Further exploration of redox active DXPS probes may provide new insights for inhibition strategies and selective probe development.