Imipenem as substrate and inhibitor of beta-lactamases.
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ABSTRACT:
SUBMITTER: Monks J
PROVIDER: S-EPMC1149301 | biostudies-other | 1988 Jul
REPOSITORIES: biostudies-other
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Project description:To determine how imipenem inhibits the class C beta-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 A. In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180 degrees compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack. This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.
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Project description:Bulgecin A, a sulphonated N-acetyl-D-glucosamine unit linked to a 4-hydroxy-5-hydroxymethylproline ring by a beta-glycosidic linkage, is a novel type of inhibitor for binuclear metallo-beta-lactamases. Using steady-state kinetic analysis with nitrocefin as the beta-lactam substrate, bulgecin A competitively inhibited the metallo-beta-lactamase BceII from Bacillus cereus in its two-zinc form, but failed to inhibit when the enzyme was in the single-zinc form. The competitive inhibition was restored by restoring the second zinc ion. The single-zinc metallo-beta-lactamase from Aeromonas veronii bv. sobria, ImiS, was not inhibited by bulgecin A. The tetrameric L1 metallo-beta-lactamase from Stenotrophomonas maltophilia was subject to partial non-competitive inhibition, which is consistent with a kinetic model in which the enzyme bound to inhibitor retains catalytic activity. Docking experiments support the conclusion that bulgecin A co-ordinates to the zinc II site in metallo-beta-lactamases via the terminal sulphonate group on the sugar moiety.
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Project description:Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed.
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Project description:SUMMARY: AmpC beta-lactamases are clinically important cephalosporinases encoded on the chromosomes of many of the Enterobacteriaceae and a few other organisms, where they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and beta-lactamase inhibitor-beta-lactam combinations. In many bacteria, AmpC enzymes are inducible and can be expressed at high levels by mutation. Overexpression confers resistance to broad-spectrum cephalosporins including cefotaxime, ceftazidime, and ceftriaxone and is a problem especially in infections due to Enterobacter aerogenes and Enterobacter cloacae, where an isolate initially susceptible to these agents may become resistant upon therapy. Transmissible plasmids have acquired genes for AmpC enzymes, which consequently can now appear in bacteria lacking or poorly expressing a chromosomal bla(AmpC) gene, such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Resistance due to plasmid-mediated AmpC enzymes is less common than extended-spectrum beta-lactamase production in most parts of the world but may be both harder to detect and broader in spectrum. AmpC enzymes encoded by both chromosomal and plasmid genes are also evolving to hydrolyze broad-spectrum cephalosporins more efficiently. Techniques to identify AmpC beta-lactamase-producing isolates are available but are still evolving and are not yet optimized for the clinical laboratory, which probably now underestimates this resistance mechanism. Carbapenems can usually be used to treat infections due to AmpC-producing bacteria, but carbapenem resistance can arise in some organisms by mutations that reduce influx (outer membrane porin loss) or enhance efflux (efflux pump activation).
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Project description:?-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete ?-lactams. However, commercially available BLIs are not effective against metallo-?-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R ? L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki* values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E*I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.
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Project description:Genes for two group 1 beta-lactamases, SRT-1 and SST-1, were sequenced. These beta-lactamases were produced by clinical isolates of Serratia marcescens, isolates GN16694 and GN19450, respectively. The resulting enzymes were 96% identical. SRT-1 hydrolyzed oxyimino cephalosporins, but SST-1 hardly hydrolyzed them. At residue 213 in the third motif, which is conserved among group 1 beta-lactamases, SRT-1 and SST-1 had Lys and Glu, respectively. By site-directed mutagenesis, the substitution of Glu by Lys at residue 213 in SST-1 resulted in an enzyme that hydrolyzed oxyimino cephalosporins.
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Project description:QPX7728 is a new ultrabroad-spectrum inhibitor of serine and metallo-beta-lactamases (MBLs) from a class of cyclic boronates that gave rise to vaborbactam. The spectrum and mechanism of beta-lactamase inhibition by QPX7728 were assessed using purified enzymes from all molecular classes. QPX7728 inhibits class A extended-spectrum beta-lactamases (ESBLs) (50% inhibitory concentration [IC50] range, 1 to 3?nM) and carbapenemases such as KPC (IC50, 2.9?±?0.4?nM) as well as class C P99 (IC50 of 22?±?8?nM) with a potency that is comparable to or higher than recently FDA-approved beta-lactamase inhibitors (BLIs) avibactam, relebactam, and vaborbactam. Unlike those other BLIs, QPX7728 is also a potent inhibitor of class D carbapenemases such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/58, IC50 range, 1 to 2?nM) as well as MBLs such as NDM-1 (IC50, 55?±?25?nM), VIM-1 (IC50, 14?±?4?nM), and IMP-1 (IC50, 610?±?70?nM). Inhibition of serine enzymes by QPX7728 is associated with progressive inactivation with a high-efficiency k 2/K ranging from 6.3?×?104 (for P99) to 9.9?×?105 M-1 s-1 (for OXA-23). This inhibition is reversible with variable stability of the QPX7728-beta-lactamase complexes with target residence time ranging from minutes to several hours: 5 to 20 min for OXA carbapenemases from A. baumannii, ?50 min for OXA-48, and 2 to 3 h for KPC and CTX-M-15. QPX7728 inhibited all tested serine enzymes at a 1:1 molar ratio. Metallo-beta-lactamases NDM, VIM, and IMP were inhibited by a competitive mechanism with fast-on-fast-off kinetics, with Ki s of 7.5?±?2.1?nM, 32?±?14?nM, and 240?±?30?nM for VIM-1, NDM-1, and IMP-1, respectively. QPX7728's ultrabroad spectrum of BLI inhibition combined with its high potency enables combinations with multiple different beta-lactam antibiotics.
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Project description:Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.
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Project description:QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor, with potent inhibition of key serine and metallo-beta-lactamases being observed in biochemical assays. Microbiological studies using characterized strains were used to provide a comprehensive characterization of the spectrum of beta-lactamase inhibition by QPX7728. The MICs of multiple antibiotics administered intravenously only (ceftazidime, piperacillin, cefepime, ceftolozane, and meropenem) and orally bioavailable antibiotics (ceftibuten, cefpodoxime, tebipenem) alone and in combination with QPX7728 (4??g/ml), as well as comparator agents, were determined against panels of laboratory strains of Pseudomonas aeruginosa and Klebsiella pneumoniae expressing over 55 diverse serine and metallo-beta-lactamases. QPX7728 significantly enhanced the potency of antibiotics against strains expressing class A extended-spectrum beta-lactamases (CTX-M, SHV, TEM, VEB, PER) and carbapenemases (KPC, SME, NMC-A, BKC-1), consistent with the beta-lactamase inhibition demonstrated in biochemical assays. It also inhibited both plasmidic (CMY, FOX, MIR, DHA) and chromosomally encoded (P99, PDC, ADC) class C beta-lactamases and class D enzymes, including carbapenemases, such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/72/58). QPX7728 is also a potent inhibitor of many class B metallo-beta-lactamases (NDM, VIM, CcrA, IMP, and GIM but not SPM or L1). Addition of QPX7728 (4??g/ml) reduced the MICs for a majority of the strains to the level observed for the control with the vector alone, indicative of complete beta-lactamase inhibition. The ultrabroad-spectrum beta-lactamase inhibition profile makes QPX7728 a viable candidate for further development.
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