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

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 β-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

Synthesis and Evaluation of Fluoroalkyl Phosphonyl Analogues of 2‑<i>C</i>‑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 <i>Plasmodium falciparum</i> and <i>Mycobacterium tuberculosis</i>, 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 <i>S</i>-monofluoro MEP analogue displays the most potent inhibitory activity against <i>Escherichia coli</i> IspD and is the best substrate for both the <i>E. coli</i> and <i>P. falciparum</i> IspD orthologues with a <i>K</i>_m approaching that of the natural substrate for the <i>E. coli</i> enzyme. This work represents a first step toward the development of phosphonyl MEP antimetabolites to modulate early isoprenoid biosynthesis in human pathogens

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

© 2019 Chen et al. 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-phos-phate (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 M, respectively) and comparable catalytic activity (kcat 45 2 min1) 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 C2-phosphonolactylThDP (PLThDP), mimicking the native pre-decarboxylation intermediate C2-lactylThDP (LThDP), and a native post-decarboxylation state with a bound enamine intermediate. The 1.94-Å-res-olution 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

The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily

El pdf del artículo es el manuscrito de autor (PMCID:PMC2813333).NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical ‘lid’ region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme–substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.I.G.G., C.E.M.S., and D.M.L. were supported by the Biotechnology and Biological Sciences Research Council through responsive mode funding (ref. B19400) and by a Core Strategic Grant to the John Innes Center. I.U. was supported by the European Union Integrated Project BIOXHIT and the Spanish MEC (grant BIO2006-14139). C.L.F.M. and C.T.W. were supported by National Institutes of Health grants F32 AI054007 and GM 20011, respectively.Peer reviewe

Crystallization and preliminary X-ray analysis of the O-carbamoyltransferase NovN from the novobiocin-biosynthetic cluster of Streptomyces spheroides

Crystals of NovN, an O-carbamoyltransferase from S. spheroides, were obtained in monoclinic and orthorhombic forms and native X-ray data were recorded to a maximum of 2.3 Å resolution

Potential Impact of Long-Acting Products on the Control of Tuberculosis: Preclinical Advancements and Translational Tools in Preventive Treatment

A key component of global tuberculosis (TB) control is the treatment of latent TB infection. The use of long-acting technologies to administer TB preventive treatment has the potential to significantly improve the delivery and impact of this important public health intervention. For example, an ideal long-acting treatment could consist of a single dose that could be administered in the clinic (ie, a "1-shot cure" for latent TB). Interest in long-acting formulations for TB preventive therapy has gained considerable traction in recent years. This article presents an overview of the specific considerations and current preclinical advancements relevant for the development of long-acting technologies of TB drugs for treatment of latent infection, including attributes of target product profiles, suitability of drugs for long-acting formulations, ongoing research efforts, and translation to clinical studies

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