Dissection of Events in the Resistance to β-Lactam Antibiotics Mediated by the Protein BlaR1 from <i>Staphylococcus aureus</i>

A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of <i>Staphylococcus aureus</i> with a central role in resistance to β-lactam antibiotics. In the absence of other <i>S. aureus</i> proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics

The Tipper–Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA)

The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-d-Ala-d-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper–Strominger hypothesis stipulates that β-lactam antibiotics mimic the acyl-d-Ala-d-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of β-lactam antibiotics, as mimics of the acyl-d-Ala-d-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different β-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA

Synthesis and NMR Characterization of (<i>Z</i>,<i>Z</i>,<i>Z</i>,<i>Z</i>,<i>E</i>,<i>E</i>,ω)-Heptaprenol

We describe a practical, multigram synthesis of (2<i>Z</i>,6<i>Z</i>,10<i>Z</i>,14<i>Z</i>,18<i>E</i>,22<i>E</i>)-3,7,11,15,19,23,27-heptamethyl-2,6,10,14,18,22,26-octacosaheptaen-1-ol [(<i>Z</i>₄,<i>E</i>₂,ω)-heptapren­ol, <b>4</b>] using the nerol-derived sulfone <b>8</b> as the key intermediate. Sulfone <b>8</b> is prepared by the literature route and is converted in five additional steps (18% yield from <b>8</b>) to (<i>Z</i>₄,<i>E</i>₂,ω)-heptaprenol <b>4</b>. The use of Eu­(hfc)₃ as an NMR shift reagent not only enabled confirmation of the structure and stereochemistry of <b>4</b>, but further enabled the structural assignment to a major side product from a failed synthetic connection. The availability by this synthesis of (<i>Z</i>₄,<i>E</i>₂,ω)-heptaprenol <b>4</b> in gram quantities will enable preparative access to key reagents for the study of the biosynthesis of the bacterial cell envelope

Revealing Cell-Surface Intramolecular Interactions in the BlaR1 Protein of Methicillin-Resistant <i>Staphylococcus aureus</i> by NMR Spectroscopy

In methicillin-resistant <i>Staphylococcus aureus</i>, β-lactam antibiotic resistance is mediated by the transmembrane protein BlaR1. The antibiotic sensor domain BlaR^S and the L2 loop of BlaR1 are on the membrane surface. We used NMR to investigate interactions between BlaR^S and a water-soluble peptide from L2. This peptide binds BlaR^S proximal to the antibiotic acylation site as an amphipathic helix. Acylation of BlaR^S by penicillin G does not disrupt binding. These results suggest a signal transduction mechanism whereby the L2 helix, partially embedded in the membrane, propagates conformational changes caused by BlaR^S acylation through the membrane via transmembrane segments, leading to antibiotic resistance

Reactions of the Three AmpD Enzymes of <i>Pseudomonas aeruginosa</i>

A group of Gram-negative bacteria, including the problematic pathogen <i>Pseudomonas aeruginosa</i>, has linked the steps in cell-wall recycling with the ability to manifest resistance to β-lactam antibiotics. A key step at the crossroads of the two events is performed by the protease AmpD, which hydrolyzes the peptide in the metabolite that influences these events. In contrast to other organisms that harbor this elaborate system, the genomic sequences of <i>P. aeruginosa</i> reveal it to have three paralogous genes for this protease, designated as <i>ampD</i>, <i>ampDh2</i>, and <i>ampDh3</i>. The recombinant gene products were purified to homogeneity, and their functions were assessed by the use of synthetic samples of three bacterial metabolites in cell-wall recycling and of three surrogates of cell-wall peptidoglycan. The results unequivocally identify AmpD as the <i>bona fide</i> recycling enzyme and AmpDh2 and AmpDh3 as enzymes involved in turnover of the bacterial cell wall itself. These findings define for the first time the events mediated by these three enzymes that lead to turnover of a key cell-wall recycling metabolite as well as the cell wall itself in its maturation

X‑ray Structure of Catenated Lytic Transglycosylase SltB1

Formation of catenanes by proteins is rare, with few known examples. We report herein the X-ray structure of a catenane dimer of lytic transglycosylase SltB1 of <i>Pseudomonas aeruginosa</i>. The enzyme is soluble and exists in the periplasmic space, where it modifies the bacterial cell wall. The catenane dimer exhibits the protein monomers in a noncovalent chain-link arrangement, whereby a stretch of 51 amino acids (to become a loop and three helices) from one monomer threads through the central opening of the structure of the partner monomer. The protein folds after threading in a manner that leaves two helices (α1 and α2) as stoppers to impart stability to the dimer structure. The symmetric embrace by the two SltB1 molecules occludes both active sites entirely, an arrangement that is sustained by six electrostatic interactions between the two monomers. In light of the observation of these structural motifs in all members of Family 3 lytic transglycosylases, catenanes might be present for those enzymes, as well. The dimeric catenane might represent a regulated form of SltB1

Regioselective Control of the S_NAr Amination of 5‑Substituted-2,4-Dichloropyrimidines Using Tertiary Amine Nucleophiles

The S_NAr reaction of 2,4-dichloropyrimidines, further substituted with an electron-withdrawing substituent at C-5, has selectivity for substitution at C-4. Here we report that tertiary amine nucleophiles show excellent C-2 selectivity. In situ N-dealkylation of an intermediate gives the product that formally corresponds to the reaction of a secondary amine nucleophile at C-2. This reaction is practical (fast under simple reaction conditions, with good generality for tertiary amine structure and moderate to excellent yields) and significantly expands access to pyrimidine structures

A Potent and Narrow-Spectrum Antibacterial against <i>Clostridioides difficile</i> Infection

Clostridioides difficile is an anaerobic Gram-positive bacterium that colonizes the gut of patients treated with broad-spectrum antibiotics. The normal gut microflora prevents C. difficile colonization; however, dysbiosis by treatment with broad-spectrum antibiotics causes recurrent C. difficile infection (CDI) in 25% of patients. There are no fully effective antibiotics for multiple recurrent CDIs. We report herein that oxadiazole antibiotics exhibit bactericidal activity against C. difficile vegetative cells. We screened a library of 75 oxadiazoles against C. difficile ATCC 43255. The findings from this collection served as the basis for the syntheses of an additional 58 analogs, which were tested against the same strain. We report a potent (MIC50 = 0.5 μg/mL and MIC90 = 1 μg/mL values for 101 C. difficile strains) and narrow-spectrum oxadiazole (3-(4-(cyclopentyloxy)­phenyl)-5-(4-nitro-1H-imidazol-2-yl)-1,2,4-oxadiazole; compound 57), which is not active against common gut bacteria or other tested organisms. Compound 57 is selectively bactericidal against C. difficile and targets cell-wall synthesis

A Potent and Narrow-Spectrum Antibacterial against <i>Clostridioides difficile</i> Infection

Clostridioides difficile is an anaerobic Gram-positive bacterium that colonizes the gut of patients treated with broad-spectrum antibiotics. The normal gut microflora prevents C. difficile colonization; however, dysbiosis by treatment with broad-spectrum antibiotics causes recurrent C. difficile infection (CDI) in 25% of patients. There are no fully effective antibiotics for multiple recurrent CDIs. We report herein that oxadiazole antibiotics exhibit bactericidal activity against C. difficile vegetative cells. We screened a library of 75 oxadiazoles against C. difficile ATCC 43255. The findings from this collection served as the basis for the syntheses of an additional 58 analogs, which were tested against the same strain. We report a potent (MIC50 = 0.5 μg/mL and MIC90 = 1 μg/mL values for 101 C. difficile strains) and narrow-spectrum oxadiazole (3-(4-(cyclopentyloxy)­phenyl)-5-(4-nitro-1H-imidazol-2-yl)-1,2,4-oxadiazole; compound 57), which is not active against common gut bacteria or other tested organisms. Compound 57 is selectively bactericidal against C. difficile and targets cell-wall synthesis

Selective Gelatinase Inhibitor Neuroprotective Agents Cross the Blood-Brain Barrier

SB-3CT, a potent and selective inhibitor of matrix metalloproteinase-2 and -9, has shown efficacy in several animal models of neurological diseases. One of the greatest challenges in the development of therapeutics for neurological diseases is the inability of drugs to cross the blood-brain barrier. A sensitive bioanalytical method based on ultraperformance liquid chromatography with multiple-reaction monitoring detection was developed to measure levels of SB-3CT, its active metabolite, the α-methyl analogue, and its <i>p</i>-hydroxy metabolite in plasma and brain. The compounds are rapidly absorbed and are readily distributed to the brain. The pharmacokinetic properties of these gelatinase inhibitors and the efficacy shown by SB-3CT in animal models of stroke, subarachnoid hemorrhage, and spinal cord injury indicate that this class of compounds holds considerable promise in the treatment of diseases of the central nervous system