Search

New antigens deriving from -lloyl and -llanyl, major and minor determinants, respectively, were produced for β-lactam antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem and aztreonam. Twenty β-lactam antigens were produced using human serum albumin and histone H1 as carrier proteins. Antigens were tested by multiplex in vitro immunoassays and evaluated based on the detection of specific IgG and IgE in the serum samples. Both major and minor determinants were appropriate antigens for detecting specific anti-β-lactam IgG in immunised rabbit sera. In a cohort of 37 allergic patients, we observed that only the minor determinants (-llanyl antigens) were suitable for determining specific anti-β-lactam IgE antibodies with high sensitivity (< 0.01 IU/mL; 24 ng/L) and specificity (100%). These findings reveal that not only the haptenisation of β-lactam antibiotics renders improved molecular recognition events when the 4-member β-lactam ring remains unmodified, but also may contribute to develop promising minor antigens suitable for detecting specific IgE-mediated allergic reactions. This will facilitate the development of sensitive and selective multiplexed in vitro tests for drug-allergy diagnoses to antibiotics cephalosporin, carbapenem and monobactam.

β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC 50 ]). MIC assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae , with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-β-lactam combinations., (Copyright © 2019 Tooke et al.)

Enhanced sampling of large number of collective variables (CVs) is inevitable in molecular dynamics (MD) simulations of complex chemical processes such as enzymatic reactions. Because of the computational overhead of hybrid quantum mechanical/molecular mechanical (QM/MM)-based MD simulations, especially together with density functional theory, predictions of reaction mechanism, and estimation of free-energy barriers have to be carried out within few tens of picoseconds. We show here that the recently developed temperature-accelerated sliced sampling method allows one to sample large number of CVs, thereby enabling us to obtain rapid convergence in free-energy estimates in QM/MM MD simulation of enzymatic reactions. Moreover, the method is shown to be efficient in exploring flat and broad free-energy basins that commonly occur in enzymatic reactions. We demonstrate this by studying deacylation and reverse acylation reactions of aztreonam drug catalyzed by a class-C β lactamase (CBL) bacterial enzyme. Mechanistic details and nature of kinetics of aztreonam hydrolysis by CBL are elaborated here. The results of this study point to characteristics of the aztreonam drug that are responsible for its slow hydrolysis.

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed., (Copyright © 2016 Vandavasi et al.)

We solved the crystal structure of the class C β-lactamase MOX-1 complexed with the inhibitor aztreonam at 1.9Å resolution. The main-chain oxygen of Ser315 interacts with the amide nitrogen of aztreonam. Surprisingly, compared to that in the structure of free MOX-1, this main-chain carboxyl changes its position significantly upon binding to aztreonam. This result indicates that the interaction between MOX-1 and β-lactams can be accompanied by conformational changes in the B3 β-strand main chain., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

New Delhi metallo-β-lactmase-1 (NDM-1) has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of β-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of β-lactam antibiotics have been detected. It could effectively hydrolyze most β-lactams (k cat/K m ratios between 0.03 to 1.28 µmol⁻¹.s⁻¹), except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM) study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

Imipenem and meropenem, members of the carbapenem class of beta-lactam antibiotics, are among the most broadly active antibiotics available for systemic use in humans. They are active against streptococci, methicillin-sensitive staphylococci, Neisseria, Haemophilus, anaerobes, and the common aerobic gram-negative nosocomial pathogens including Pseudomonas. Resistance to imipenem and meropenem may emerge during treatment of P. aeruginosa infections, as has occurred with other beta-lactam agents; Stenotrophomonas maltophilia is typically resistant to both imipenem and meropenem. Like the penicillins, the carbapenems have inhibitory activity against enterococci. In general, the in vitro activity of imipenem against aerobic gram-positive cocci is somewhat greater than that of meropenem, whereas the in vitro activity of meropenem against aerobic gram-negative bacilli is somewhat greater than that of imipenem. Daily dosages may range from 0.5 to 1 g every 6 to 8 hours in patients with normal renal function; the daily dose of meropenem, however, can be safely increased to 6 g. Infusion-related nausea and vomiting, as well as seizures, which have been the main toxic effects of imipenem, occur no more frequently during treatment with meropenem than during treatment with other beta-lactam antibiotics. The carbapenems should be considered for treatment of mixed bacterial infections and aerobic gram-negative bacteria that are not susceptible to other beta-lactam agents. Indiscriminate use of these drugs will promote resistance to them. Aztreonam, the first marketed monobactam, has activity against most aerobic gram-negative bacilli including P. aeruginosa. The drug is not nephrotoxic, is weakly immunogenic, and has not been associated with disorders of coagulation. Aztreonam may be administered intramuscularly or intravenously; the primary route of elimination is urinary excretion. In patients with normal renal function, the recommended dosing interval is every 8 hours. Patients with renal impairment require dosage adjustment. Aztreonam is used primarily as an alternative to aminoglycosides and for the treatment of aerobic gram-negative infections. It is often used in combination therapy for mixed aerobic and anaerobic infections. Approved indications for its use include infections of the urinary tract or lower respiratory tract, intra-abdominal and gynecologic infections, septicemia, and cutaneous infections caused by susceptible organisms. Concurrent initial therapy with other antimicrobial agents is recommended before the causative organism has been determined in patients who are seriously ill or at risk for gram-positive or anaerobic infection.

The catalytic effect of various buffer systems (citrates, acetates, phosphates, borates and carbonates) on the degradation of aztreonam and nocardicin A in aqueous solution was studied at 35 degrees C and a constant ionic strength of 0.5 mol.dm-3 over a pH range of 3.50 to 10.50. The observed degradation rates, obtained by measuring the remaining intact antibiotic, were shown to follow pseudo-first-order kinetics with regard to antibiotic concentrations and to be influenced by general acid and general base catalysis. The changes in the concentration of intact beta-lactam antibiotic in the solutions were established by reverse-phase HPLC with UV-detection. In general the buffer systems employed in the kinetic studies showed a very weak catalytic effect on the degradation of aztreonam and nocardicin A. The pH-rate profiles for these antibiotics showed degradation minimums at pH 5.38 and 6.13, respectively. Aztreonam is slightly more reactive with hydrogen ions than nocardicin A and is much more reactive with hydroxide ions. In comparison with other beta-lactamic antibiotics, aztreonam and nocardicin A are much more stable in aqueous solution, except for aztreonam in a base solution, which is just as unstable as penicillins and cephalosporins. The Arrhenius activation energies were determined for aztreonam and nocardicin A at pH's 4.23, 6.59 and 8.60.