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Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled--making LPS synthesis a promising target for developing new antibiotics. Escherichia coli adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapBcyto). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapBcyto/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapBcyto also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Pseudomonas aeruginosa. Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH. [ABSTRACT FROM AUTHOR]

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc amidase that catalyzes the second step of the biosynthesis of lipid A, which is an outer membrane essential structural component of Gram-negative bacteria. Inhibitors of this enzyme can be attributed to two main categories, non-hydroxamate and hydroxamate inhibitors, with the latter being the most effective given the chelation of Zn2+ in the active site. Compounds containing diacetylene or acetylene tails and the sulfonic head, as well as oxazoline derivatives of hydroxamic acids, are among the LpxC inhibitors with the most profound antibacterial activity. The present article describes the synthesis of novel functional derivatives of hydroxamic acids—bioisosteric to oxazoline inhibitors—containing 1,2,4- and 1,3,4-oxadiazole cores and studies of their cytotoxicity, antibacterial activity, and antibiotic potentiation. Some of the hydroxamic acids we obtained (9c, 9d, 23a, 23c, 30b, 36) showed significant potentiation in nalidixic acid, rifampicin, and kanamycin against the growth of laboratory-strain Escherichia coli MG1655. Two lead compounds (9c, 9d) significantly reduced Pseudomonas aeruginosa ATCC 27853 growth in the presence of nalidixic acid and rifampicin. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria, such as Escherichia coli, are characterized by an asymmetric outer membrane (OM) with lipopolysaccharide (LPS) located in the outer leaflet and phospholipids facing the inner leaflet. E. coli recruits LPS assembly proteins LapB, LapC and LapD in concert with FtsH protease to ensure a balanced biosynthesis of LPS and phospholipids. We recently reported that bacteria either lacking the periplasmic domain of the essential LapC protein (lapC190) or in the absence of LapD exhibit an elevated degradation of LpxC, which catalyzes the first committed step in LPS biosynthesis. To further understand the functions of LapC and LapD in regulating LPS biosynthesis, we show that the overproduction of the intact LapD suppresses the temperature sensitivity (Ts) of lapC190, but not when either its N-terminal transmembrane anchor or specific conserved amino acids in the C-terminal domain are mutated. Moreover, overexpression of srrA, marA, yceJ and yfgM genes can rescue the Ts phenotype of lapC190 bacteria by restoring LpxC amounts. We further show that MarA-mediated suppression requires the expression of mla genes, whose products participate in the maintenance of OM asymmetry, and the SrrA-mediated suppression requires the presence of cardiolipin synthase A. [ABSTRACT FROM AUTHOR]

Salmonella typhi is 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 typhi and improve typhoid fever treatment. [ABSTRACT FROM AUTHOR]

Human deaths caused by Gram-negative bacteria keep rising due to the multidrug resistance (MDR) phenomenon. Therefore, it is a priority to develop novel antibiotics with different mechanisms of action. Several bacterial zinc metalloenzymes are becoming attractive targets since they do not show any similarities with the human endogenous zinc-metalloproteinases. In the last decades, there has been an increasing interest from both industry and academia in developing new inhibitors against those enzymes involved in lipid A biosynthesis, and bacteria nutrition and sporulation, e.g., UDP-[3-O-(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC), thermolysin (TLN), and pseudolysin (PLN). Nevertheless, targeting these bacterial enzymes is harder than expected and the lack of good clinical candidates suggests that more effort is needed. This review gives an overview of bacterial zinc metalloenzyme inhibitors that have been synthesized so far, highlighting the structural features essential for inhibitory activity and the structure–activity relationships. Our discussion may stimulate and help further studies on bacterial zinc metalloenzyme inhibitors as possible novel antibacterial drugs. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) has emerged as one of the global threats to human health in the 21st century. Drug discovery of inhibitors against novel targets rather than conventional bacterial targets has been considered an inevitable strategy for the growing threat of AMR infections. In this study, we applied quantitative structureactivity relationship (QSAR) modeling to the LpxC inhibitors to predict the inhibitory activity. In addition, we performed various cheminformatics analysis consisting of the exploration of the chemical space, identification of chemotypes, performing structure-activity landscape and activity cliffs as well as construction of the StructureActivity Similarity (SAS) map. We built a total of 24 QSAR classification models using PubChem and MACCS fingerprint with 12 various machine learning algorithms. The best model with PubChem fingerprint is the Extremely Gradient Boost model (accuracy on the training set: 0.937; accuracy on the 10-fold cross-validation set: 0.795; accuracy on the test set: 0.799). Furthermore, it was found that the best model using the MACCS fingerprint was the Random Forest model (accuracy on the training set: 0.955; accuracy on the 10-fold cross-validation set: 0.803; accuracy on the test set: 0.785). In addition, we have identified eight consensus activity cliff generators that are highly informative for further SAR investigations. It is hoped that findings presented herein can provide guidance for further lead optimization of LpxC inhibitors. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as Escherichia coli. Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved is unknown. Examination of proteins co-purifying with LapB identified LapD as a new partner. The purification of LapD reveals that it forms a complex with several proteins involved in LPS and phospholipid biosynthesis, including FtsH-LapA/B and Fab enzymes. Loss of LapD causes a reduction in LpxC amounts and vancomycin sensitivity, which can be restored by mutations that stabilize LpxC (mutations in lapB, ftsH and lpxC genes), revealing that LapD acts upstream of LapB-FtsH in regulating LpxC amounts. Interestingly, LapD absence results in the substantial retention of LPS in the inner membranes and synthetic lethality when either the lauroyl or the myristoyl acyl transferase is absent, which can be overcome by single-amino acid suppressor mutations in LPS flippase MsbA, suggesting LPS translocation defects in ΔlapD bacteria. Several genes whose products are involved in cell envelope homeostasis, including clsA, waaC, tig and micA, become essential in LapD's absence. Furthermore, the overproduction of acyl carrier protein AcpP or transcriptional factors DksA, SrrA can overcome certain defects of the LapD-lacking strain. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Uridine diphosphate-3-O-(hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a key enzyme involved in the biosynthesis of lipid A, an essential building block, for the construction and assembly of the outer membrane (OM) of Gram-negative bacteria. The enzyme is highly conserved in almost all Gram-negative bacteria and hence has emerged as a promising target for drug discovery in the fight against multi-drug resistant Gram-negative infections. Since the first nanomolar LpxC inhibitor, L-161,240, an oxazoline-based hydroxamate, the two-decade-long ongoing search has provided valuable information regarding essential features necessary for inhibition. Although the design and structure optimization for arriving at the most efficacious inhibitor of this enzyme has made good use of different heterocyclic moieties, the use of carbohydrate scaffold is scant. This review briefly covers the advancement and progress made in LpxC inhibition. The field awaits the use of potential associated with carbohydrate-based scaffolds for LpxC inhibition and the discovery of anti-bacterial agents against Gram-negative infections. [Display omitted] • LpxC is a promising target in combatting Gram-negative bacteria. • This review briefly covers the advancement and progress achieved in LpxC inhibition. • This review highlights the use of carbohydrate-based scaffolds for LpxC inhibition. [ABSTRACT FROM AUTHOR]

The outer membrane (OM) is a defining feature of Gram-negative bacteria that serves as a permeability barrier and provides rigidity to the cell. Critical to OM function is establishing and maintaining an asymmetrical bilayer structure with phospholipids in the inner leaflet and the complex glycolipid lipopolysaccharide (LPS) in the outer leaflet. Cells ensure this asymmetry by regulating the biogenesis of lipid A, the conserved and essential anchor of LPS. Here we review the consequences of disrupting the regulatory components that control lipid A biogenesis, focusing on the rate-limiting step performed by LpxC. Dissection of these processes provides critical insights into bacterial physiology and potential new targets for antibiotics able to overcome rapidly spreading resistance mechanisms. Lipopolysaccharide (LPS) is found in most Gram-negative bacteria and is critical for outer membrane (OM) barrier function and maintaining cell rigidity. Gram-negative bacteria precisely regulate LPS levels as imbalances are lethal: too much LPS appears to disrupt inner-membrane integrity while too little weakens the OM. In Escherichia coli , LPS levels are regulated by the YciM/FtsH protease complex, which degrades the enzyme that catalyzes the first committed step in lipid A biogenesis, LpxC. Factors in multiple cellular compartments regulate activity of the YciM/FtsH protease complex in response to changes in LPS levels, including the essential inner-membrane protein YejM, the outer-membrane phospholipase PldA, and the lipid A biosynthetic intermediate lipid A disaccharide in the cytoplasm. [ABSTRACT FROM AUTHOR]

The antibacterial effect of volatile compounds in millets and cereal by‐products was investigated. Hexane extracts of rice bran (RB), corn cob (CC), green (GM), and yellow millets (YM) showed antibacterial activities against Escherichia coli ATCC 35150, Bacillus cereus ATCC 14579, Helicobacter pylori MH179991, and Staphylococcus aureus ATCC 13150 with minimum inhibitory concentration (MIC) ranging from 2.5 to 5 mg/ml. Gas chromatography‐mass spectrometry analysis revealed a total of 101 compounds in all extracts. Furthermore, the mode of action of the major bioactive compounds in extracts was tested against Lipoprotein X complex (LpxC) and four chained structured binding protein of Bacterial type II topoisomerase (4PLB) in silico. Docking analysis showed moderate to potent inhibition against receptor proteins (4PLB and LpxC) with a docking score of −7.52, −8.89, and −9.89 Kcal/mol for 3,5‐dimethylbenzaldehyde, cyclododecane, di‐tert‐butylbenzene, respectively. GM, YM, CC, and RB hexane extracts possess antibacterial potential that could be developed as antimicrobial agents. Practical Applications: Cereals and their by‐products represent an abundant and low‐cost resource for value‐added functional food. They may be considered alternative new sources of antimicrobial agents to tackle a challenge of antimicrobial‐resistance. Findings from this work provide insight into how volatile bioactive compounds inhibit bacterial pathogens by targeting 4PLB and LpxC as evident in the computational study. Further studies to confirm the antibacterial potency of these compounds in vivo is warranted. [ABSTRACT FROM AUTHOR]

Introduction: Treatment failures of standard regimens and new strains egression are due to the augmented drug resistance conundrum. These confounding factors now became the drug designers spotlight to implement therapeutics against Helicobacter pylori strains and to safeguard infected victims with devoid of adverse drug reactions. Thereby, to navigate the chemical space for medicine, paramount vital drug target opting considerations should be imperative. The study is therefore aimed to develop potent therapeutic variants against an insightful extrapolative, common target LpxC as a follow-up to previous studies. Methods: We explored the relationships between existing inhibitors and novel leads at the scaffold level in an appropriate conformational plasticity for lead-optimization campaign. Hierarchical-clustering and shape-based screening against an in-house library of > 21 million compounds resulted in panel of 11,000 compounds. Rigid-receptor docking through virtual screening cascade, quantum-polarized-ligand, induced-fit dockings, post-docking processes and system stability assessments were performed. Results: After docking experiments, an enrichment performance unveiled seven ranked actives better binding efficiencies with Zinc-binding potency than substrate and in-actives (decoy-set) with ROC (1.0) and area under accumulation curve (0.90) metrics. Physics-based membrane permeability accompanied ADME/T predictions and long-range dynamic simulations of 250 ns chemical time have depicted good passive diffusion with no toxicity of leads and sustained consistency of lead1-LpxC in the physiological milieu respectively. Conclusions: In the study, as these static outcomes obtained from this approach competed with the substrate and existing ligands in binding affinity estimations as well as positively correlated from different aspects of predictions, which could facilitate promiscuous new chemical entities against H. pylori. [ABSTRACT FROM AUTHOR]

To model substrate binding to zinc containing enzymes, Zn(Tp ∗ )Cl (Tp ∗  = tris(3,5-dimethyl-1-pyrazolyl)borate) was investigated as an inexpensive and easily prepared target. Both acetyl hydroxamic acid and acetic acid, common zinc binding moieties, were found to bind zinc in our model system and the resulting complexes were fully characterized. The hydroxamate complex was found to be both thermodynamically and kinetically favored in comparison to the acetate complex, in keeping with previous models, demonstrating the utility of this system in enzymatic mimicry. [ABSTRACT FROM AUTHOR]

Copper-catalyzed azide-alkyne cycloadditions (CuAAC or click chemistry) are convenient methods to easily couple various pharmacophores or bioactive molecules. A new series of 1,2,3-triazole-linked nucleoside-amino acid conjugates have been designed and synthesized in 57-76% yields using CuAAC. The azido group was introduced on the 50-position of uridine or the acyclic analogue using the tosyl-azide exchange method and alkylated serine or proparylglycine was the alkyne. Modeling studies of the conjugates in the active site of LpxC indicate they have promise as antibacterial agents. [ABSTRACT FROM AUTHOR]

As an important target for the development of novel antibiotics, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) has been widely studied. Pyridone methylsulfone hydroxamate (PMH) compounds can effectively inhibit the catalytic activity of LpxC. In this work, the three-dimensional quantitative structure-activity relationships of PMH inhibitors were explored and models with good predictive ability were established using comparative molecular field analysis and comparative molecular similarity index analysis methods. The effect of PMH inhibitors’ electrostatic potential on the inhibitory ability of Pseudomonas aeruginosa LpxC (PaLpxC) is revealed at the molecular level via molecular electrostatic potential analyses. Then, two molecular dynamics simulations for the PaLpxC and PaLpxC-inhibitor systems were also performed respectively to investigate the key residues of PaLpxC hydrolase binding to water molecules. The results indicate that orderly alternative water molecules can form stable hydrogen bonds with M62, E77, T190, and H264 in the catalytic center, and tetracoordinate to zinc ion along with H78, H237, and D241. It was found that the conformational transition space of PaLpxC changes after association with PMH inhibitors through free energy landscape and cluster analyses. Finally, a possible inhibitory mechanism of PMH inhibitors was proposed, based on our molecular simulation. This paper will provide a theoretical basis for the molecular design of LpxC-targeting antibiotics. [ABSTRACT FROM AUTHOR]

The emergence and spread of multidrug-resistant (MDR) Gram negative bacteria presents a serious threat for public health. Novel antimicrobials that could overcome the resistance problems are urgently needed. UDP-3-O-( R -3-hydroxymyristol)- N -acetylglucosamine deacetylase (LpxC) is a cytosolic zinc-based deacetylase that catalyzes the first committed step in the biosynthesis of lipid A, which is essential for the survival of Gram-negative bacteria. Our efforts toward the discovery of novel LpxC inhibitors are presented herein. [ABSTRACT FROM AUTHOR]

We attempted to optimize sulfonamide-based non-alkyne LpxC inhibitors by focusing on improvements in enzyme inhibitory and antibacterial activity. It was discovered that inhibitors possessing 2-aryl benzofuran as a hydrophobe exhibited good activity. In particular, compound 21 displayed impressive antibacterial activity ( E. coli MIC = 0.063 μg/mL, K. pneumoniae MIC = 0.5 μg/mL, and P. aeruginosa MIC = 0.5 μg/mL), and is a promising lead for further exploration as an antibacterial agent. [ABSTRACT FROM AUTHOR]

Uridine diphosphate-3-O-(hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metalloenzyme with zinc ions as cofactors and is a key enzyme in the essential structural outer membrane lipid A synthesis commitment step of gram-negative bacteria. As LpxC is extremely homologous among different Gram-negative bacteria, it is conserved in almost all gram-negative bacteria, which makes LpxC a promising target. LpxC inhibitors have been reported extensively in recent years, such as PF-5081090 and CHIR-090 were found to have broad-spectrum antibiotic activity against P. aeruginosa and E. coli. They are mainly classified into hydroxamate inhibitors and non-hydroxamate inhibitors based on their structure, but no LpxC inhibitors have been marketed due to safety and activity issues. This review, therefore, focuses on small molecule inhibitors of LpxC against gram-negative pathogenic bacteria and covers recent advances in LpxC inhibitors, focusing on their structural optimization process, structure-activity relationships, and future directions, with the aim of providing ideas for the development of LpxC inhibitors and clinical research. [Display omitted] • Summary of structure, function and mechanisms of LpxC. • The analysis of structure and structure-activity relationship of LpxC inhibitors. • The current progress and novel strategies of LpxC small molecule inhibitors. • The development and future of LpxC inhibitors. [ABSTRACT FROM AUTHOR]

Microbial infections are becoming resistant to traditional antibiotics. As novel resistance mechanisms are developed and disseminated across the world, our ability to treat the most common infectious diseases is becoming increasingly compromised. As existing antibiotics are losing their effectiveness, especially treatment of bacterial infections, is difficult. In order to combat this issue, it is of utmost importance to identify novel pharmacological targets or antibiotics. LpxC, a zinc-dependent metalloamidase that catalyzes the committed step in the biosynthesis of lipid A (endotoxin) in bacteria, is a prime candidate for drug/therapeutic target. So far, the rate-limiting metallo-amidase LpxC has been the most-targeted macromolecule in the Raetz pathway. This is because it is important for the growth of these bacterial infections. This review showcases on the research done to develop efficient drugs in this area before and after the 2015. [ABSTRACT FROM AUTHOR]

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3- O -( R -3-hydroxymyristoyl)- N -acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB , a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design. [ABSTRACT FROM AUTHOR]

Abstract: Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs. [Copyright &y& Elsevier]

Abstract: Lipid A is an essential component of the Gram negative outer membrane, which protects the bacterium from attack of many antibiotics. The Lipid A biosynthesis pathway is essential for Gram negative bacterial growth and is unique to these bacteria. The first committed step in Lipid A biosynthesis is catalysis by LpxC, a zinc dependent deacetylase. We show the design of an LpxC inhibitor utilizing a robust model which directed efficient design of picomolar inhibitors. Analysis of physiochemical properties drove design to focus on an optimal lipophilicity profile. Further structure based design took advantage of a conserved water network over the active site, and with the optimal lipophilicity profile, led to an improved LpxC inhibitor with in vivo activity against wild type Pseudomonas aeruginosa. [Copyright &y& Elsevier]

Abstract: Proteolysis of regulatory proteins or key enzymes of biosynthetic pathways is a universal mechanism to rapidly adjust the cellular proteome to particular environmental needs. Among the five energy-dependent AAA+ proteases in Escherichia coli, FtsH is the only essential protease. Moreover, FtsH is unique owing to its anchoring to the inner membrane. This review describes the structural and functional properties of FtsH. With regard to its role in cellular quality control and regulatory circuits, cytoplasmic and membrane substrates of the FtsH protease are depicted and mechanisms of FtsH-dependent proteolysis are discussed. This article is part of a Special Issue entitled: AAA ATPases: structure and function. [Copyright &y& Elsevier]

Abstract: Antibiotic resistant hospital acquired infections are on the rise, creating an urgent need for novel bactericidal drugs. Enzymes involved in lipopolysaccharide (LPS) biosynthesis are attractive antibacterial targets since LPS is the major structural component of the outer membrane of Gram-negative bacteria. Lipid A is an essential hydrophobic anchor of LPS and the first committed step in lipid A biosynthesis is catalyzed by a unique zinc dependent metalloamidase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC is an attractive Gram-negative only target that has been chemically validated by potent bactericidal hydroxamate inhibitors that work by coordination of the enzyme’s catalytic zinc ion. An exploratory chemistry effort focused on expanding the SAR around hydroxamic acid zinc-binding ‘warheads’ lead to the identification of novel compounds with enzyme potency and antibacterial activity similar to CHIR-090. [Copyright &y& Elsevier]

Abstract: Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group. [ABSTRACT FROM AUTHOR]

Abstract: Control of cellular processes by regulated proteolysis is conserved among all organisms. FtsH, the only membrane-anchored AAA protease in bacteria, fulfills a variety of regulatory functions. This review focuses on soluble FtsH substrates in Escherichia coli and in other bacteria and outlines emerging substrate recognition principles. [Copyright &y& Elsevier]

The outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, is essential for their viability. Lipopolysaccharide (LPS) constitutes the major component of OM, providing the permeability barrier, and a tight balance exists between LPS and phospholipids amounts as both of these essential components use a common metabolic precursor. Hence, checkpoints are in place, right from the regulation of the first committed step in LPS biosynthesis mediated by LpxC through its turnover by FtsH and HslUV proteases in coordination with LPS assembly factors LapB and LapC. After the synthesis of LPS on the inner leaflet of the inner membrane (IM), LPS is flipped by the IM-located essential ATP-dependent transporter to the periplasmic face of IM, where it is picked up by the LPS transport complex spanning all three components of the cell envelope for its delivery to OM. MsbA exerts its intrinsic hydrocarbon ruler function as another checkpoint to transport hexa-acylated LPS as compared to underacylated LPS. Additional checkpoints in LPS assembly are: LapB-assisted coupling of LPS synthesis and translocation; cardiolipin presence when LPS is underacylated; the recruitment of RfaH transcriptional factor ensuring the transcription of LPS core biosynthetic genes; and the regulated incorporation of non-stoichiometric modifications, controlled by the stress-responsive RpoE sigma factor, small RNAs and two-component systems. [ABSTRACT FROM AUTHOR]

Abstract: The membrane-anchored FtsH protease is essential in Escherichia coli as it adjusts the cellular amount of LpxC, the key enzyme in lipopolysaccharide (LPS) biosynthesis. Both accumulation and depletion of LpxC are toxic to E. coli. By continuous proteolysis of LpxC, FtsH maintains a low concentration of LpxC and, hence, the proper equilibrium between LPS and phospholipids. The C terminus of LpxC is required for turnover. By adding this tail to glutathione-S-transferase (GST) we show that it is necessary but not sufficient for FtsH-mediated degradation. A detailed mutational analysis revealed six non-polar residues in the C terminus of LpxC that are critical for degradation. Alteration of the C-terminal AVLA motif towards the SsrA-like sequence ALAA directed LpxC to other cellular proteases reinforcing the importance of the C-terminal tail for targeting to FtsH. Short C-terminal truncations stabilized LpxC. Most mutations in the C terminus of LpxC left its enzymatic activity intact as was shown by growth assays, microscopy and 2-keto-3-deoxyoctonate (KDO) determination. The critical length of the turnover element was defined by internal deletions. A C-terminal tail of about 20 amino acids length is required for proteolysis of LpxC by FtsH. [Copyright &y& Elsevier]

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We used a differential pathway analyses approach (based on KEGG data) to identify essential genes from Pseudomonas aeruginosa. Our approach identified 214 unique enzymes in P. aeruginosa that may be potential drug targets and can be considered for rational drug design. About 40% of these putative targets have been reported as essential by transposon mutagenesis data elsewhere. Homology model for one of the proteins (LpxC) is presented as a case study and can be explored for in silico docking with suitable inhibitors. This approach is a step towards facilitating the search for new antibiotics. [ABSTRACT FROM AUTHOR]

The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens. Heterologous expression of lipid A modification enzymes may enable the development of new vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Various electronic properties of structurally diverse synthetic LpxC inhibitors containing oxazoline, aroylserine and thiazoline rings were calculated and correlated with biological activity. These electronic features include the magnitude and locations of 3-dimensional molecular electrostatic potentials, hydrogen bond acceptor/donor density, lowest unoccupied molecular orbital, and highest occupied molecular orbital. Strong correlation of these stereo-electronic properties with LpxC inhibitory potency reveals the potential pharmacophoric features of specific LpxC inhibitors. Thus, these pharmacophoric features of LpxC inhibitors based on electronic and surface analysis could be successfully exploited for designing more potent LpxC inhibitors. [Copyright &y& Elsevier]

Abstract: A class of metalloenzymes, known as zinc hydrolases, catalyze a variety of hydrolytic reactions on many different substrates in important metabolic pathways. Deacetylation is an example of one of the types of reactions catalyzed by zinc hydrolases. The biological importance of the reactions catalyzed by many zinc hydrolases, including zinc-dependent deacetylases, has made these enzymes pharmaceutical targets for the development of inhibitors and, therefore, a clear understanding of the mechanisms of these enzymes is warranted. This review focuses on the current understanding of the mechanisms catalyzed by various zinc-dependent deacetylases and, in particular, the reaction mechanism catalyzed by the enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, also known as LpxC. In general, the zinc–water functions as the nucleophile with zinc stabilization of the tetrahedral intermediate and general-acid–base catalysis (GABC) provided by enzyme residue(s). Two types of GABC mechanisms have been identified, one that uses a single bifunctional GABC and another that uses a GABC pair. [Copyright &y& Elsevier]

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO2(His)2 site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins. [Copyright &y& Elsevier]

To identify the physiological factors that limit the growth of Escherichia coli K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire waa locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IVA derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ(gmhD-waaA) identified two independent single-amino-acid substitutions—P50S and R310S—in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene clsA was found to be essential for the growth of ΔlpxLMP, ΔlpxL, ΔwaaA, and Δ(gmhD-waaA) bacteria, with a conditional lethal phenotype of Δ(clsA lpxM), which could be overcome by suppressor mutations in MsbA. Suppressor mutations basS A20D or basR G53V, causing a constitutive incorporation of phosphoethanolamine (P-EtN) in the lipid A, could abolish the Ca++ sensitivity of Δ(waaC eptB), thereby compensating for P-EtN absence on the second Kdo. A single-amino-acid OppA S273G substitution is shown to overcome the synthetic lethality of Δ(waaC surA) bacteria, consistent with the chaperone-like function of the OppA oligopeptide-binding protein. Furthermore, overexpression of GcvB sRNA was found to repress the accumulation of LpxC and suppress the lethality of LapAB absence. Thus, this study identifies new and limiting factors in regulating LPS biosynthesis. [ABSTRACT FROM AUTHOR]

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways. [ABSTRACT FROM AUTHOR]

New drugs with novel antibacterial targets for Gram-negative bacterial pathogens are desperately needed. The protein LpxC is a vital enzyme for the biosynthesis of lipid A, an outer membrane component of Gram-negative bacterial pathogens. The ACHN-975 molecule has high enzymatic inhibitory capacity against the infectious diseases, which are caused by multidrug-resistant bacteria, but clinical research was halted because of its inflammatory response in previous studies. In this work, the structure of the recombinant UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase from Aquifex aeolicus in complex with ACHN-975 was determined to a resolution at 1.21 Å. According to the solved complex structure, ACHN-975 was docked into the AaLpxC's active site, which occupied the site of AaLpxC substrate. Hydroxamate group of ACHN-975 forms five-valenced coordination with resides His74, His226, Asp230, and the long chain part of ACHN-975 containing the rigid alkynyl groups docked in further to interact with the hydrophobic area of AaLpxC. We employed isothermal titration calorimetry for the measurement of affinity between AaLpxC mutants and ACHN-975, and the results manifest the key residues (His74, Thr179, Tyr212, His226, Asp230 and His253) for interaction. The determined AaLpxC crystal structure in complex with ACHN-975 is expected to serve as a guidance and basis for the design and optimization of molecular structures of ACHN-975 analogues to develop novel drug candidates against Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the development of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with existing drugs has been earnestly desired. UDP-3- O -acyl- N -acetylglucosamine deacetylase (LpxC) is a drug target that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result, a development candidate, TP0586532 , was created that is effective against carbapenem-resistant Klebsiella pneumoniae and does not pose a cardiovascular risk. [ABSTRACT FROM AUTHOR]

UDP-3- O -(R -3-hydroxyacyl)- N -acetylglucosamine deacetylase (LpxC), the zinc metalloenzyme catalyzing the first committed step of lipid A biosynthesis in Gram-negative bacteria, has been a target for antibacterial drug discovery for many years. All inhibitor chemotypes reaching an advanced preclinical stage and clinical phase 1 have contained terminal hydroxamic acid, and none have been successfully advanced due, in part, to safety concerns, including hemodynamic effects. We hypothesized that the safety of LpxC inhibitors could be improved by replacing the terminal hydroxamic acid with a different zinc-binding group. After choosing an N -hydroxyformamide zinc-binding group, we investigated the structure-activity relationship of each part of the inhibitor scaffold with respect to Pseudomonas aeruginosa and Escherichia coli LpxC binding affinity, in vitro antibacterial potency and pharmacological properties. We identified a novel, potency-enhancing hydrophobic binding interaction for an LpxC inhibitor. We demonstrated in vivo efficacy of one compound in a neutropenic mouse E. coli infection model. Another compound was tested in a rat hemodynamic assay and was found to have a hypotensive effect. This result demonstrated that replacing the terminal hydroxamic acid with a different zinc-binding group was insufficient to avoid this previously recognized safety issue with LpxC inhibitors. [ABSTRACT FROM AUTHOR]

This study evaluated the in vitro activity of KHP-3757 (a novel LpxC inhibitor) and comparator agents against recent, geographically diverse, Pseudomonas aeruginosa isolates from a global surveillance program as well as molecularly characterized extended-spectrum-β-lactamase-positive, metallo-β-lactamase-positive, and colistin-resistant strains. KHP-3757 (MIC 50/90 , 0.25/0.5 mg/L; 97.4% inhibited at ≤0.5 mg/L) demonstrated potent in vitro activity based on MIC 90 values against P. aeruginosa isolates including extended-spectrum-β-lactamase-positive, metallo-β-lactamase-positive, and colistin-resistant strains outperforming other comparator agents. [ABSTRACT FROM AUTHOR]

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of ΔlapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts. [ABSTRACT FROM AUTHOR]

LpxC is a deacetylase that catalyzes the first committed step of lipid A biosynthesis in Escherichia coli. LpxC competes for a common precursor, R-3-hydroxymyristoyl-UDP-GlcNAc, with FabZ, whose dehydratase activity catalyzes the first committed step of phospholipid biosynthesis. To maintain the optimum flow of the common precursor to these two competing pathways, the LpxC level is controlled by FtsH/YciM-mediated proteolysis. It is not known whether this complex or another protein senses the status of lipid A synthesis to control LpxC proteolysis. The work carried out in this study began with a novel mutation, yejM1163, which causes hypersensitivity to large antibiotics such as vancomycin and erythromycin. Isolates resistant to these antibiotics carried suppressor mutations in the ftsH and yciM genes. Western blot analysis showed a dramatically reduced LpxC level in the yejM1163 background, while the presence of ftsH or yciM suppressor mutations restored LpxC levels to different degrees. Based on these observations, it is proposed that YejM is a sensor of lipid A synthesis and controls LpxC levels by modulating the activity of the FtsH/YciM complex. The truncation of the periplasmic domain in the YejM1163 protein causes unregulated proteolysis of LpxC, thus diverting a greater pool of R-3-hydroxymyristoyl-UDP-GlcNAc toward phospholipid synthesis. This imbalance in lipid synthesis perturbs the outer membrane permeability barrier, causing hypersensitivity toward vancomycin and erythromycin. yejM1163 suppressor mutations in ftsH and yciM lower the proteolytic activity toward LpxC, thus restoring lipid homeostasis and the outer membrane permeability barrier. IMPORTANCE Lipid homeostasis is critical for proper envelope functions. The level of LpxC, which catalyzes the first committed step of lipopolysaccharide (LPS) synthesis, is controlled by an essential protease complex comprised of FtsH and YciM. Work carried out here suggests YejM, an essential envelope protein, plays a central role in sensing the state of LPS synthesis and controls LpxC levels by regulating the activity of FtsH/YciM. All four essential proteins are attractive targets of therapeutic development. [ABSTRACT FROM AUTHOR]

The outer membranes of Gram-negative bacteria provide a permeability barrier to antibiotics and other harmful chemicals. The integrity of this barrier relies on the maintenance of the lipid asymmetry of the outer membrane, and studies of suppressors of a decades-old mutant reveal that YejM plays a key regulatory role and provide a model for the maintenance of this asymmetry. [ABSTRACT FROM AUTHOR]

Distinguishing feature of the outer membrane (OM) of Gram-negative bacteria is its asymmetry due to the presence of lipopolysaccharide (LPS) in the outer leaflet of the OM and phospholipids in the inner leaflet. Recent studies have revealed the existence of regulatory controls that ensure a balanced biosynthesis of LPS and phospholipids, both of which are essential for bacterial viability. LPS provides the essential permeability barrier function and act as a major virulence determinant. In Escherichia coli, more than 100 genes are required for LPS synthesis, its assembly at inner leaflet of the inner membrane (IM), extraction from the IM, translocation to the OM, and in its structural alterations in response to various environmental and stress signals. Although LPS are highly heterogeneous, they share common structural elements defining their most conserved hydrophobic lipid A part to which a core polysaccharide is attached, which is further extended in smooth bacteria by O-antigen. Defects or any imbalance in LPS biosynthesis cause major cellular defects, which elicit envelope responsive signal transduction controlled by RpoE sigma factor and two-component systems (TCS). RpoE regulon members and specific TCSs, including their non-coding arm, regulate incorporation of non-stoichiometric modifications of LPS, contributing to LPS heterogeneity and impacting antibiotic resistance. [ABSTRACT FROM AUTHOR]