ABSTRACT
Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of ß-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the ß-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications likely enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulator mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance lysis of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.
Subject(s)
Bacterial Proteins/metabolism , Carbapenems/pharmacology , Drug Tolerance , Enterobacter cloacae/drug effects , Enterobacter cloacae/metabolism , Lipopolysaccharides/metabolism , Anti-Bacterial Agents/pharmacology , Antimicrobial Peptides/pharmacology , Cell Membrane/drug effects , Cell Membrane/metabolism , Colistin/pharmacology , Enterobacter cloacae/genetics , Gene Expression Regulation , Histidine Kinase/antagonists & inhibitors , Humans , Lipid A/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microbial Sensitivity TestsABSTRACT
BACKGROUND: Retinoic acid (RA) controls diverse physiological functions including weight regulation and energy metabolism. It has been reported that mice lacking ALDH1A1, one of the aldehyde dehydrogenases (ALDH) that synthesize RA, are healthy and resistant to weight gain, raising the possibility that inhibiting this enzyme might treat obesity. We previously demonstrated that treatment with a pan-ALDH1A enzyme inhibitor, WIN18446, suppressed weight gain in mice fed a high-fat diet (HFD), but caused increased hepatic lipidosis and reversible male infertility. METHODS: A series of piperazine compounds that inhibited ALDH1A1 were identified and their inhibitory activity was characterized in vitro using purified recombinant enzymes and cell-based assay systems. One potent compound, FSI-TN42 (N42) was examined for its oral bioavailability and pharmacodynamic effects. In addition, its effect on weight gain was investigated by daily oral administration to C57BL/6 male mice receiving a HFD, and compared with mice receiving WIN18446 or vehicle alone (n = 6/group, 200 mg compound/kg body weight) for 5 weeks. Body weights were measured weekly, and a glucose tolerance test was performed after 4 weeks of treatment. Tissues were collected to determine changes in adipose weight, hepatic lipidosis, retinoid metabolism, and expression of genes associated with RA and lipid metabolism. RESULTS: N42 irreversibly binds and inhibits ALDH1A1 in vitro with a low nM IC50 and 800-fold specificity for ALDH1A1 compared to ALDH1A2. Daily oral administration of N42 significantly suppressed weight gain (P < 0.05) and reduced visceral adiposity (p < 0.05) in mice fed a HFD without the hepatic lipidosis observed with WIN18446 treatment. CONCLUSIONS: We developed a potent and specific inhibitor of ALDH1A1 that suppressed weight gain in mice fed a HFD. These findings demonstrate that inhibition of ALDH1A1 is a feasible target for drug development to treat and/or prevent obesity.
Subject(s)
Aldehyde Dehydrogenase 1 Family/antagonists & inhibitors , Obesity/metabolism , Piperazines/pharmacology , Retinal Dehydrogenase/antagonists & inhibitors , Weight Gain/drug effects , Adipose Tissue/drug effects , Administration, Oral , Animals , Diet, High-Fat , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Piperazines/administration & dosage , Piperazines/chemistryABSTRACT
Histidine kinases of bacterial two-component systems are promising antibacterial targets. Despite their varied, numerous roles, enzymes in the histidine kinase superfamily share a catalytic core that may be exploited to inhibit multiple histidine kinases simultaneously. Characterized by the Bergerat fold, the features of the histidine kinase ATP-binding domain are not found in serine/threonine and tyrosine kinases. However, because each kinase family binds the same ATP substrate, we sought to determine if published serine/threonine and tyrosine kinase inhibitors contained scaffolds that would also inhibit histidine kinases. Using select assays, 222 inhibitors from the Roche Published Kinase Set were screened for binding, deactivation, and aggregation of histidine kinases. Not only do the results of our screen support the distinctions between ATP-binding domains of different kinase families, but the lead molecule identified also presents inspiration for further histidine kinase inhibitor development.
Subject(s)
Histidine Kinase/metabolism , Protein Kinase Inhibitors/chemistry , Serine/chemistry , Threonine/chemistry , Adenosine Triphosphate/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Binding Sites , Catalytic Domain , Histidine Kinase/antagonists & inhibitors , Molecular Docking Simulation , Protein Kinase Inhibitors/metabolism , Serine/metabolism , Thermotoga maritima/enzymology , Threonine/metabolismABSTRACT
Heparin is a sulfated glycosaminoglycan that is widely used as an anticoagulant. It is typically extracted from porcine or bovine sources to yield a heterogeneous mixture that varies both in molecular weight and in degree of sulfation. This heterogeneity, coupled with concern for contamination, has led to widespread interest in developing safer alternatives. Described herein are sulfated bacteriophage Qß virus-like particles (VLPs) that elicit heparin-like anticoagulant activity. Sulfate groups were appended to the VLP by synthesis of single- and triple-sulfated ligands that also contained azide groups. Following conversion of VLP surface lysine groups to alkynes, the sulfated ligands were attached to the VLP via copper-catalyzed azide-alkyne cycloaddition (CuAAC). MALDI-MS analysis of the intermediate alkyne VLP indicated that the majority of the coat proteins contained 5-7 of the alkyne linkers; similar analysis of the intermediate alkyne particles conjugated to a fluorescein azide suggest that nearly the same number of attachment points (3-6) are modified via CuAAC. Analysis by SDS-PAGE with fluorescent staining indicated altered migration patterns for the various constructs: compared to the wild-type nanoparticle, the modified coat proteins appeared to migrate farther toward the positive pole in the gel, with coat proteins displaying the triple-sulfated ligand migrating significantly farther. Clotting activity analyzed by activated partial thrombin time (APTT) assay showed that the sulfated particles were able to perturb coagulation, with VLPs displaying the triple-sulfated ligand approximately as effective as heparin on a per mole basis; this activity could be partially reversed by protamine. ELISA experiments to assess the response of the complement system to the VLPs indicate that sulfating the particles may reduce complement activation.
Subject(s)
Allolevivirus/chemistry , Anticoagulants/chemistry , Anticoagulants/pharmacology , Heparin/chemistry , Heparin/pharmacology , Nanoparticles/chemistry , Sulfates/chemistry , Alkynes/chemistry , Azides/chemistry , Blood Coagulation/drug effects , Capsid Proteins/chemistry , Complement Activation/drug effects , Copper/chemistry , Cycloaddition Reaction , Humans , Ligands , Models, Molecular , Partial Thromboplastin Time , Protein ConformationABSTRACT
Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.
ABSTRACT
With the rise in antimicrobial resistance and the dearth of effective strategies to combat this threat, the development of novel therapies is of utmost importance. Targeting of bacterial signaling through their the two-component systems (TCSs) may be a viable strategy. TCSs are comprised of a sensory histidine kinase (HK), of which a bacterium can have up to 160 distinct proteins, and a cognate response regulator (RR). The TCSs are generally non-essential for life, but control many virulence and antibiotic-resistance mechanisms. This, along with their absence in animals makes the TCSs an attractive target for antimicrobial therapy, whether as a stand-alone treatments or adjuvants for existing therapies. This review focuses on progress in the development of inhibitors that target the HK ATP-binding domain. Because this domain is highly conserved, it may be feasible to disrupt multiple TCSs within a single organism to increase effectiveness and reduce pressure for the evolution of resistance.