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In a fragment-based approach using NMR spectroscopy, benzyloxyacetohydroxamic acid-derived inhibitors of the bacterial deacetylase LpxC bearing a substituent to target the uridine diphosphate-binding site of the enzyme were developed. By appending privileged fragments via a suitable linker, potent LpxC inhibitors with promising antibacterial activities could be obtained, like the one-digit nanomolar LpxC inhibitor (S)-13j[Ki(EcLpxC C63A) = 9.5 nM; Ki(PaLpxC): 5.6 nM]. To rationalize the observed structure–activity relationships, molecular docking and molecular dynamics studies were performed. Initial in vitro absorption–distribution–metabolism–excretion–toxicity (ADMET) studies of the most potent compounds have paved the way for multiparameter optimization of our newly developed isoserine-based amides.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a promising drug target in Gram-negative bacteria. Previously, we described a correlation between the residence time of inhibitors on Pseudomonas aeruginosaLpxC (paLpxC) and the post-antibiotic effect (PAE) caused by the inhibitors on the growth of P. aeruginosa. Given that drugs with prolonged activity following compound removal may have advantages in dosing regimens, we have explored the structure–kinetic relationship for paLpxC inhibition by analogues of the pyridone methylsulfone PF5081090(1) originally developed by Pfizer. Several analogues have longer residence times on paLpxC than 1(41 min) including PT913, which has a residence time of 124 min. PT913also has a PAE of 4 h, extending the original correlation observed between residence time and PAE. Collectively, the studies provide a platform for the rational modulation of paLpxC inhibitor residence time and the potential development of antibacterial agents that cause prolonged suppression of bacterial growth.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation. A series was evolved from a fragment where a glycine moiety complexes zinc, which achieved low nanomolar potency in an enzyme functional assay but poor antibacterial activity on cell cultures. A second series was based on a fragment that chelated zinc through an imidazole moiety. Structure-guided design led to a 2-(1S-hydroxyethyl)-imidazole derivative exhibiting low nanomolar inhibition of LpxC and a minimum inhibitory concentration (MIC) of 4 μg/mL against Pseudomonas aeruginosa, which is little affected by the presence of albumin.

UDP-3-O-((R)-3-hydroxymyristoyl)-N-glucosamine deacetylase (LpxC) is as an attractive target for the discovery and development of novel antibacterial drugs to address the critical medical need created by multidrug resistant Gram-negative bacteria. By using a scaffold hopping approach on a known family of methylsulfone hydroxamate LpxC inhibitors, several hit series eliciting potent antibacterial activities against Enterobacteriaceae and Pseudomonas aeruginosawere identified. Subsequent hit-to-lead optimization, using cocrystal structures of inhibitors bound to Pseudomonas aeruginosaLpxC as guides, resulted in the discovery of multiple chemical series based on (i) isoindolin-1-ones, (ii) 4,5-dihydro-6H-thieno[2,3-c]pyrrol-6-ones, and (iii) 1,2-dihydro-3H-pyrrolo[1,2-c]imidazole-3-ones. Synthetic methods, antibacterial activities and relative binding affinities, as well as physicochemical properties that allowed compound prioritization are presented. Finally, in vivo properties of lead molecules which belong to the most promising pyrrolo-imidazolone series, such as 18d, are discussed.

Antimicrobial resistance (AMR) is currently a global health concern, mostly caused by microorganisms like bacteria, viruses, parasites, and fungi that acquire resistance to antimicrobial drugs. Salmonellais responsible for a variety of diseases but mainly cause typhoid. The primary concern is the rise in resistance in both non-typhoid and typhoid strains of this species. To address this issue, it is necessary to identify novel targets and strategies for the development of new antibacterial drugs. Lipid A, a strong bacterial endotoxin that modulates the immune system in human, is a key component of the virulence factor generated during the salmonellainfection. Lipid A is synthesized in case of Gram-negative bacteria by cascade of nine enzyme pathway. The second step in case of Lipid A biosynthesis, catalysed by LpxC, a Zn+dependent metallo-amidase considered as rate limiting step. In this manuscript we have used protein-ligand interaction fingerprint (PLIF)–derived pharmacophore models to screen small molecules (natural products library from Zinc database, Asinex database, Thiophene analogues) against Salmonella typhiLpxC (StLpxC). Further top hit molecules were subjected to MD-simulation and ADMET studies. We identified three optimal compounds, s1_dl_mseq2, s1_ll_mseq2, and s2_ll_mseq8, that exhibit strong binding affinity towards the LpxC active site.

LpxC inhibitors were optimized starting from lead compounds with limited efficacy and solubility and with the goal to provide new options for the treatment of serious infections caused by Gram-negative pathogens in hospital settings. To enable the development of an aqueous formulation for intravenous administration of the drug at high dose, improvements in both solubility and antibacterial activity in vivo were prioritized early on. This lead optimization program resulted in the discovery of compounds such as 13and 30, which exhibited high solubility and potent efficacy against Gram-negative pathogens in animal infection models.

Keywords: Durham; State:North Carolina; United States; North and Central America; Translational Medicine; Health and Medicine EN Durham State:North Carolina United States North and Central America Translational Medicine Health and Medicine 6126 6126 1 09/11/23 20230915 NES 230915 2023 SEP 15 (NewsRx) -- By a News Reporter-Staff News Editor at Health & Medicine Week -- Data detailed on Health and Medicine - Translational Medicine have been presented. The development of LpxC-targeting antibiotics toward clinical therapeutics has been hindered by the limited antibiotic profile of reported non-hydroxamate inhibitors and unexpected cardiovascular toxicity observed in certain hydroxamate and non-hydroxamate-based inhibitors." Durham, State:North Carolina, United States, North and Central America, Translational Medicine, Health and Medicine. [Extracted from the article]

Infections caused by pathogenic bacteria are a major health concern throughout the world. There is a great need to develop novel antibacterial agents with new mechanisms of action. Lipopolysaccharides (LPS) are the main component of the outer membrane of Gram-negative bacteria, serving as a permeability barrier, which protects the bacteria from many antibiotics. The UDP-3-O-(R-3- hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC), a Zn2+-dependent enzyme, catalyzes the first irreversible step of the biosynthesis of lipid A, the hydrophobic membrane anchor of LPS being essential for cell viability. Additionally, it shares no sequence or structural homology with any mammalian proteins. Therefore, it may become a novel target for the new drugs against Gram-negative bacteria. Thus, research on LpxC inhibitors as new antibacterial agents has become an attractive field in the development of the novel antibiotic therapy of Gram-negative bacteria. In this review, we will summarize the recent progress in the structure and catalytic mechanism of LpxC and the research and development of LpxC inhibitors.

Salmonella typhiis an infectious bacteria that causes typhoid fever and poses a significant risk to human health. The emergence of antibiotic resistance has become a growing concern in the management of this disease. In this work, a structure-based drug design approach was used to identify inhibitors for zinc-dependent metalloamidase LpxC, the enzyme responsible for the biosynthesis of lipid A. Using an in silico approach (virtual screening, docking, and molecular dynamics (MD) simulations), from a library of 59,000 indole derivatives, we were able to identify promising lead molecules with high binding affinity to the LpxC. Of these, five molecules (compound 435 (CID: 12253558), compound 436 (CID: 122514279), compound 1812 (CID: 90797680), compound 2584 (CID: 57056726), and compound 2545 (CID: 59897361)) have passed all the filtering criteria. This finding was verified by molecular dynamics (MD) simulation as well as post-dynamics free energy calculations. The five compounds that have been identified have shown the most promise compared to other compounds that are already recognized. To further validate the positive outcome of this study, experimental validation and optimization are necessary. These lead compounds may help to develop new antibiotics for antibiotic-resistant Salmonella typhiand improve typhoid fever treatment.

This report summarizes the identification and synthesis of novel LpxC inhibitors aided by computational methods that leveraged numerous crystal structures. This effort led to the identification of oxazolidinone and isoxazoline inhibitors with potent in vitro activity against P. aeruginosaand other Gram-negative bacteria. Representative compound 13fdemonstrated efficacy against P. aeruginosain a mouse neutropenic thigh infection model. The antibacterial activity against K. pneumoniaecould be potentiated by Gram-positive antibiotics rifampicin (RIF) and vancomycin (VAN) in both in vitro and in vivo models.

Keywords for this news article include: Bangkok, Thailand, Asia, Cyborgs, Drugs and Therapies, Emerging Technologies, Machine Learning, Mahidol University. Keywords: Bangkok; Thailand; Asia; Cyborgs; Drugs and Therapies; Emerging Technologies; Machine Learning EN Bangkok Thailand Asia Cyborgs Drugs and Therapies Emerging Technologies Machine Learning 2731 2731 1 10/30/23 20231103 NES 231103 2023 NOV 3 (NewsRx) -- By a News Reporter-Staff News Editor at Drug Week -- Research findings on Machine Learning are discussed in a new report. [Extracted from the article]

Keywords: Tamil Nadu; India; Asia; Drugs and Therapies; Enterobacteriaceae; Gammaproteobacteria; Gram-Negative Bacteria; Gram-Negative Facultatively Anaerobic Rods; Molecular Dynamics; Physics; Proteobacteria; Salmonella; Salmonella enterica; Salmonella typhi EN Tamil Nadu India Asia Drugs and Therapies Enterobacteriaceae Gammaproteobacteria Gram-Negative Bacteria Gram-Negative Facultatively Anaerobic Rods Molecular Dynamics Physics Proteobacteria Salmonella Salmonella enterica Salmonella typhi 1523 1523 1 08/28/23 20230901 NES 230901 2023 SEP 1 (NewsRx) -- By a News Reporter-Staff News Editor at Drug Week -- Current study results on Gram-Negative Bacteria - Salmonella typhi have been published. Keywords for this news article include: Tamil Nadu, India, Asia, Drugs and Therapies, Enterobacteriaceae, Gammaproteobacteria, Gram-Negative Bacteria, Gram-Negative Facultatively Anaerobic Rods, Molecular Dynamics, Physics, Proteobacteria, Salmonella, Salmonella enterica, Salmonella typhi, Vellore Institute of Technology. [Extracted from the article]

ABSTRACTIntroduction: The Zn2+-dependent deacetylase LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising target for the development of antibiotics selectively combating Gram-negative pathogens. Researchers from industry and academia have synthesized structurally diverse LpxC inhibitors, exhibiting different LpxC inhibitory and antibacterial activities.Areas covered: A brief introduction into the structure and function of LpxC, showing its suitability as antibacterial target, along with the structures of several reported LpxC inhibitors, is given. The article reviews patents (reported between 2010 and 2016) and related research publications on novel small-molecule LpxC inhibitors. Emphasis is placed on structure-activity relationships within the reported series of LpxC inhibitors.Expert opinion: The performed analysis of patents revealed that the current search for novel LpxC inhibitors is focused on small molecules, sharing common structural features like a Zn2+-chelating group as well as a highly lipophilic side-chain. However, despite the promising preclinical data of many of the reported compounds, besides the recently withdrawn clinical candidate ACHN-975, no other LpxC inhibitor has entered clinical trials. The lack of clinical candidates might be related with undesired effects caused by the common structural elements of the LpxC inhibitors.

Background: Bacterial resistance is currently a significant global public health problem. Acinetobacter baumanniihas been ranked in the list of the World Health Organization as the most critical and priority pathogen for which new antibiotics are urgently needed. In this context, computational methods play a central role in the modern drug discovery process. The purpose of the current study was to identify new potential therapeutic molecules to neutralize MDR A. baumanniibacteria. Methods: A total of 3686 proteins retrieved from the A. baumanniiproteome were subjected to subtractive proteomic analysis to narrow down the spectrum of drug targets. The SWISS‐MODEL server was used to perform a 3D homology model of the selected target protein. The SAVES server was used to evaluate the overall quality of the model. A dataset of 74500 analogues retrieved from the PubChem database was docked with LpxC using the AutoDock software. Results: In this study, we predicted a putative new inhibitor for the Lpxc enzyme of A. baumannii. The LpxC enzyme was selected as the most appropriate drug target for A. baumannii. According to the virtual screening results, N‐[(2S)‐3‐amino‐1‐(hydroxyamino)‐1‐oxopropan‐2‐yl]‐4‐(4‐bromophenyl) benzamide (CS250) could be a promising drug candidate targeting the LpxC enzyme. This molecule shows polar interactions with six amino acids and non‐polar interactions with eight other residues. In vitroexperimental validation was performed through the inhibition assay. Conclusion: To the best of our knowledge, this is the first study that suggests CS250 as a promising inhibitory molecule that can be exploited to target this gram‐negative pathogen.

The bacterial enzyme UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC), catalyzing the first committed step of lipid A biosynthesis, represents a promising target in the development of novel antibiotics against Gram-negative bacteria. Structure, catalytic reaction mechanism and regulation of the Zn2+-dependent metalloamidase have been intensively investigated. The enzyme is required for growth and viability of Gram-negative bacteria, displays no sequence homology with any mammalian protein, but is highly conserved in Gram-negative bacteria, thus permitting the development of Gram-negative selective antibacterial agents with limited off-target effects. Several smallmolecule LpxC inhibitors have been developed, like the substrate analog TU-514 (12a), the aryloxazoline L-161,240 (13w), the sulfonamide BB-78485 (23a), the N-aroyl-L-threonine derivative CHIR-090 (24a), the sulfone-containing pyridone LpxC-3 (43e), and the uridine-based inhibitor 1-68A (47a), displaying diverse inhibitory and antibacterial activities. Most of these compounds share a Zn2+-binding hydroxamate moiety attached to a structural element addressing the hydrophobic tunnel or the UDP binding site. The butadiynyl derivative ACHN-975 (28) is the first LpxC inhibitor entering clinical trials.

ABSTRACTLpxC inhibitors have generally shown poor in vitroactivity against Acinetobacter baumannii. We show that the LpxC inhibitor PF-5081090 inhibits lipid A biosynthesis, as determined by silver staining and measurements of endotoxin levels, and significantly increases cell permeability. The presence of PF-5081090 at 32 mg/liter increased susceptibility to rifampin, vancomycin, azithromycin, imipenem, and amikacin but had no effect on susceptibility to ciprofloxacin and tigecycline. Potentiating existing antibiotics with LpxC inhibitors may represent an alternative treatment strategy for multidrug-resistant A. baumannii.

"In some embodiments, the at least one pharmaceutically acceptable excipient is a co-solvent, oil, surfactant, complexing agent, a solubilizing polymer, a P-gp modulator, a buffering agent, or a combination thereof. The pharmaceutical composition of claim 40, wherein the cellulose polymer excipient comprises cellulose acetate phthalate, carboxymethylcellulose sodium, hydroxypropylcellulose acetate succinate, hydroxypropyl methylcellulose 606(HPMC 606), or hydroxypropyl methylcellulose phthalate (HP-55). In some embodiments, the cellulose polymer excipient comprises cellulose acetate phthalate, carboxymethylcellulose sodium, hydroxypropylcellulose acetate succinate, hydroxypropyl methylcellulose 606 (HPMC 606), or hydroxypropyl methylcellulose phthalate (HP-55). "In some embodiments, the at least one pharmaceutically acceptable excipient is a co-solvent, oil, surfactant, complexing agent, a solubilizing polymer, a P-gp modulator, a buffering agent, or a combination thereof. [Extracted from the article]

Two series of novel LpxC inhibitors with hydrophilic terminus have been synthesized and their in vitroantibacterial activities against Escherichia coliand Pseudomonas aeruginosawere evaluated. Especially, compounds 22band cexhibited comparable antibacterial activities to CHIR-090 and better metabolic stability than CHIR-090 and LPC-011 in liver microsomes.

Current studies continue to show that UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetyl glucosamine deacetylase (LpxC) inhibitors such as pyridone methylsulfone hydroxymate 1 and 2a (LpxC-2) downregulate the lethal effects of sepsis initiated by multi-drug resistant Gram-negative bacteria (MDRGNB) by curtailing lipopolysaccharide (LPS) synthesis in murine models. Sepsis initiated by MDRGNB is a leading cause of shock and systemic inflammatory response syndrome (SIRS) in intensive care unit (ICU) patients. To date however, the biological effects of LpxC-2 and related molecules in companion and production animals remain largely unexplored in vivoand are therefore unknown. Such studies would be greatly informative in the expectation of LpxC-2 progressing to human clinical trials. Mechanistic studies to interrogate this novel antibiotic candidate in realistic and clinically applicable large animal models of veterinary importance are sorely lacking. To be relevant, the physiology of the chosen animal models should closely match that of humans such as ovine or porcine, or even better, non-human primate based studies, as they are more genetically similar to humans than murine models. If discovered to have subtle or negligible side effects, LpxC-2 could have a future role in the treatment and management of MDRGNB-induced infections that lead to sepsis in both animals and humans. More research is indicated on LpxC-2 use in many veterinary species, as data remains scarce.

AbstractCurrent studies continue to show that UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetyl glucosamine deacetylase (LpxC) inhibitors such as pyridone methylsulfone hydroxymate 1 and 2a (LpxC-2) downregulate the lethal effects of sepsis initiated by multi-drug resistant Gram-negative bacteria (MDRGNB) by curtailing lipopolysaccharide (LPS) synthesis in murine models. Sepsis initiated by MDRGNB is a leading cause of shock and systemic inflammatory response syndrome (SIRS) in intensive care unit (ICU) patients. To date however, the biological effects of LpxC-2 and related molecules in companion and production animals remain largely unexplored in vivoand are therefore unknown. Such studies would be greatly informative in the expectation of LpxC-2 progressing to human clinical trials. Mechanistic studies to interrogate this novel antibiotic candidate in realistic and clinically applicable large animal models of veterinary importance are sorely lacking. To be relevant, the physiology of the chosen animal models should closely match that of humans such as ovine or porcine, or even better, non-human primate based studies, as they are more genetically similar to humans than murine models. If discovered to have subtle or negligible side effects, LpxC-2 could have a future role in the treatment and management of MDRGNB-induced infections that lead to sepsis in both animals and humans. More research is indicated on LpxC-2 use in many veterinary species, as data remains scarce.

Leptospira interrogans, a Gram-negative bacterial pathogen is the main cause of human leptospirosis. Lipid A is a highly immunoreactive endotoxic center of lipopolysaccharide (LPS) that anchors LPS into the outer membrane of Leptospira. Discovery of compounds inhibiting lipid-A biosynthetic pathway would be promising for dissolving the structural integrity of membrane leading to cell lysis and death of Leptospira. LpxC, a unique enzyme of lipid-A biosynthetic pathway was identified as common drug target of Leptospira. Herein, homology modeling, docking, and molecular dynamics (MD) simulations were employed to discover potential inhibitors of LpxC. A reliable tertiary structure of LpxC in complex with inhibitor BB-78485 was constructed in Modeller 9v8. A data-set of BB-78485 structural analogs were docked with LpxC in Maestro v9.2 virtual screening workflow, which implements three stage Glide docking protocol. Twelve lead molecules with better XP Gscore compared to BB-78485 were proposed as potential inhibitors of LpxC. Para-(benzoyl)-phenylalanine – that showed lowest XP Gscore (−10.35 kcal/mol) – was predicted to have best binding affinity towards LpxC. MD simulations were performed for LpxC and para-(benzoyl)-phenylalanine docking complex in Desmond v3.0. Trajectory analysis showed the docking complex and inter-molecular interactions was stable throughout the entire production part of MD simulations. The results indicate para-(benzoyl)-phenylalanine as a potent drug molecule against leptospirosis.An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:10

The rapid increase of health-threatening infections by Gram-negative pathogens along with the emergence of multidrugresistant bacterial strains demands the development of novel antibiotics directed against the previously unexploited targets. One of the promising targets in Gram-negative bacteria is the zinc-dependent metalloamidase, UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine deacetylase (LpxC). LpxC catalyzes the first committed, second overall step in the biosynthetic pathway of lipid A. Thus, research on LpxC inhibitors as antibacterial agents has become an attractive field in the development of the novel antibiotic therapy of Gram-negative bacteria. In this review, we will summarize the recent progress in the studies on the structure, catalytic mechanism and regulation of LpxC and the current development of LpxC inhibitors.

Testing P. aeruginosa efflux pump mutants showed that the LpxC inhibitor CHIR-090 is a substrate for MexAB-OprM, MexCD-OprJ, and MexEF-OprN. Utilizing P. aeruginosa PAO1 with a chromosomal mexC::luxCDABE fusion, luminescent mutants arose on medium containing 4 μg/ml CHIR-090, indicating upregulation of MexCD-OprJ. These mutants were less susceptible to CHIR-090 (MIC, 4 μg/ml) and had mutations in the mexCD-oprJ repressor gene nfxB. Nonluminescent mutants (MIC, 4 μg/ml) that had mutations in the mexAB-oprM regulator gene mexR were also observed. Plating the clinical isolate K2153 on 4 μg/ml CHIR-090 selected mutants with alterations in mexS (immediately upstream of mexT), which upregulates MexEF-OprN. A mutant altered in the putative1ribosomal binding site (RBS) upstream of lpxC and overexpressing LpxC was selected on a related LpxC inhibitor and exhibited reduced susceptibility to CHIR-090. Overexpression of LpxC from a plasmid reduced susceptibility to CHIR-090, and introduction of the altered RBS in this construct further increased expression of LpxC and decreased susceptibility to CHIR-090. Using a mutS (hypermutator) strain, a mutant with an altered lpxC target gene (LpxC L18V) was also selected. Purified LpxC L18V had activity similar to that of wild-type LpxC in an in vitro assay but had reduced inhibition by CHIR-090. Finally, an additional class of mutant, typified by an extreme growth defect, was identified. These mutants had mutations in fabG, indicating that alteration in fatty acid synthesis conferred resistance to LpxC inhibitors. Passaging experiments showed progressive decreases in susceptibility to CHIR-090. Therefore, P. aeruginosa can employ several strategies to reduce susceptibility to CHIR-090 in vitro.

ABSTRACTTesting P. aeruginosaefflux pump mutants showed that the LpxC inhibitor CHIR-090 is a substrate for MexAB-OprM, MexCD-OprJ, and MexEF-OprN. Utilizing P. aeruginosaPAO1 with a chromosomal mexC::luxCDABEfusion, luminescent mutants arose on medium containing 4 μg/ml CHIR-090, indicating upregulation of MexCD-OprJ. These mutants were less susceptible to CHIR-090 (MIC, 4 μg/ml) and had mutations in the mexCD-oprJrepressor gene nfxB. Nonluminescent mutants (MIC, 4 μg/ml) that had mutations in the mexAB-oprMregulator gene mexRwere also observed. Plating the clinical isolate K2153 on 4 μg/ml CHIR-090 selected mutants with alterations in mexS(immediately upstream of mexT), which upregulates MexEF-OprN. A mutant altered in the putative1ribosomal binding site (RBS) upstream of lpxCand overexpressing LpxC was selected on a related LpxC inhibitor and exhibited reduced susceptibility to CHIR-090. Overexpression of LpxC from a plasmid reduced susceptibility to CHIR-090, and introduction of the altered RBS in this construct further increased expression of LpxC and decreased susceptibility to CHIR-090. Using a mutS(hypermutator) strain, a mutant with an altered lpxCtarget gene (LpxC L18V) was also selected. Purified LpxC L18V had activity similar to that of wild-type LpxC in an in vitroassay but had reduced inhibition by CHIR-090. Finally, an additional class of mutant, typified by an extreme growth defect, was identified. These mutants had mutations in fabG, indicating that alteration in fatty acid synthesis conferred resistance to LpxC inhibitors. Passaging experiments showed progressive decreases in susceptibility to CHIR-090. Therefore, P. aeruginosacan employ several strategies to reduce susceptibility to CHIR-090 in vitro.

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire™ mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z′ factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coliwith reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 μg/mL). The results show that mass spectrometry–based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

Background: Human infections due to Gram-negative bacteria cause significant morbidity and mortality. Identification of new strategies, molecular targets, and agents for the treatment of Gram-negative bacterial infections are needed urgently. Lipid A is a necessary component of the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. The zinc-dependent hydrolase UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) involved in the first committed step in the biosynthetic pathway of lipid A has no sequence homology to any known mammalian enzymes and has emerged as an attractive Gram-negative antibacterial molecular target. Most previously described LpxC inhibitors contain a hydroxamic acid, which can lead to low specificity vs. other metal-dependent enzymes and can consequently result in unwanted side effects. Objective: This review examines a new reported class of nonhydroxamic LpxC inhibitors for the treatment of Gram-negative infections. Methods: The new class of inhibitor is compared with several previously reported LpxC inhibitors. Conclusion: The LpxC inhibitors disclosed in PCT application WO 2008027466 contain hydantoins in place of the hydroxamic acids commonly found in most previously described inhibitors. These molecules could represent a means of treating Gram-negative infections via a more selective inhibition of LpxC.