ABSTRACT
Background: In a previous study, the efficacy and safety of sulbactam-durlobactam vs colistin for the treatment of patients with carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRABC) infections were evaluated in a randomized controlled phase 3 trial. Both arms were dosed on a background of imipenem-cilastatin to treat coinfecting gram-negative pathogens. Thirty-six percent of infections in the primary efficacy population were polymicrobial. Methods: A subset analysis was performed to compare clinical and microbiological outcomes at test of cure (7 ± 2 days after the last dose) for patients with monomicrobial and polymicrobial CRABC infections. Minimal inhibitory concentrations of antibiotics against baseline isolates were determined by broth microdilution according to Clinical and Laboratory Standards Institute methodology. Results: Clinical cure, 28-day all-cause mortality, and microbiological outcomes were similar for patients in the sulbactam-durlobactam treatment arm with monomicrobial or polymicrobial A baumannii-calcoaceticus infections. Patients in the colistin arm with monomicrobial CRABC infections had higher mortality rates with worse clinical and microbiological outcomes as compared with those with polymicrobial infections. For patients who received sulbactam-durlobactam, imipenem susceptibility of coinfecting gram-negative pathogens trended with clinical benefit for patients with polymicrobial A baumannii-calcoaceticus infections. When tested in vitro, durlobactam restored imipenem susceptibility to the majority of coinfecting gram-negative pathogens from the sulbactam-durlobactam arm. This phenotype appeared to be related to the clinical outcome in 13 of 15 evaluable cases. Conclusions: These results suggest that the use of sulbactam-durlobactam plus a carbapenem could be an effective approach to treat polymicrobial infections that include CRABC, but additional clinical data are needed to demonstrate efficacy.
ABSTRACT
Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe infections that are difficult to treat due to pre-existing antibiotic resistance. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat serious infections caused by Acinetobacter, including multidrug- and carbapenem-resistant strains. In a recent global surveillance study of 5,032 ABC clinical isolates collected from 2016 to 2021, less than 2% of ABC isolates had SUL-DUR MIC values >4 µg/mL. Molecular characterization of these isolates confirmed the primary drivers of resistance are metallo-ß-lactamases or penicillin-binding protein 3 (PBP3) mutations, as previously described. In addition, this study shows that certain common PBP3 variants, such as A515V, are insufficient to confer sulbactam resistance and that the efflux of durlobactam by AdeIJK is likely to play a role in a subset of strains.
Subject(s)
Acinetobacter baumannii , Sulbactam , Sulbactam/pharmacology , Sulbactam/therapeutic use , Anti-Bacterial Agents/therapeutic use , Azabicyclo Compounds/pharmacology , beta-Lactamase Inhibitors/pharmacology , beta-Lactamase Inhibitors/therapeutic use , Monobactams , Microbial Sensitivity TestsABSTRACT
The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Design , Drug Resistance, Multiple, Bacterial , Gram-Negative Bacteria/drug effects , Animals , Anti-Bacterial Agents/chemistry , Aza Compounds/chemistry , Aza Compounds/pharmacology , Cyclooctanes/chemistry , Cyclooctanes/pharmacology , Female , Mice , Mice, Inbred BALB C , Molecular Structure , Penicillin-Binding Proteins/antagonists & inhibitors , Pseudomonas aeruginosa/drug effects , beta-LactamasesABSTRACT
Durlobactam is a new member of the diazabicyclooctane class of ß-lactamase inhibitors with broad spectrum activity against Ambler class A, C, and D serine ß-lactamases. Sulbactam is a first generation ß-lactamase inhibitor with activity limited to a subset of class A enzymes that also has direct-acting antibacterial activity against Acinetobacter spp. The latter feature is due to sulbactam's ability to inhibit certain penicillin-binding proteins, essential enzymes involved in bacterial cell wall synthesis in this pathogen. Because sulbactam is also susceptible to cleavage by numerous ß-lactamases, its clinical utility for the treatment of contemporary Acinetobacter infections is quite limited. However, when combined with durlobactam, the activity of sulbactam is effectively restored against these notoriously multidrug-resistant strains. This sulbactam-durlobactam combination is currently in late-stage development for the treatment of Acinectobacter infections, including those caused by carbapenem-resistant isolates, for which there is a high unmet medical need. The following mini-review summarizes the molecular drivers of efficacy of this combination against this troublesome pathogen, with an emphasis on the biochemical features of each partner.
ABSTRACT
In this study, we sought to determine whether an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Nucleoside-Phosphate Kinase/metabolism , Rhodanine/metabolism , Anti-Bacterial Agents/chemistry , DNA, Bacterial/genetics , Drug Discovery , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Genome, Bacterial , Microbial Sensitivity Tests , Microbial Viability , Models, Molecular , Molecular Structure , Nucleoside-Phosphate Kinase/antagonists & inhibitors , Nucleoside-Phosphate Kinase/genetics , Protein ConformationABSTRACT
Mutations in KPC-2 and KPC-3 ß-lactamase can confer resistance to the ß-lactam/ß-lactamase inhibitor antibacterial intravenous drug combination ceftazidime-avibactam, introduced in 2015. Avibactam was the first of the diazabicyclooctane class of non-ß-lactam ß-lactamase inhibitors to be approved for clinical use. The orally bioavailable prodrug ETX0282 of the diazabicyclooctane ß-lactamase inhibitor ETX1317 is in clinical development in combination with the oral ß-lactam prodrug cefpodoxime proxetil for use against complicated urinary tract infections. We investigated the effects of 3 ceftazidime-avibactam resistance mutations in KPC-3 (V240G, D179Y, and D179Y/T243M) on the ability of ETX1317 to overcome KPC-3-induced cefpodoxime resistance. Isogenic Escherichia coli strains, each expressing the wild-type or a mutant KPC-3 at similar levels, retained susceptibility to cefpodoxime-ETX1317 (1:2) with essentially identical minimal inhibitory concentrations of 0.125-0.25 µg/mL cefpodoxime. The KPC-3 mutations had little or no effect on the kinact/Ki values for inhibition by each of 3 diazabicyclooctanes: avibactam, durlobactam (ETX2514), and ETX1317. The KM values for hydrolysis of cefpodoxime were similar for all 4 variants, but the kcat values of the D179Y and D179Y/T243M variants were much lower than those of the wild-type and V240G mutant enzymes. All 4 KPC-3 variants formed stable, reversibly covalent complexes with ETX1317, but dissociation of ETX1317 was much slower from the D179Y and D179Y/T243M mutants than from the wild-type and V240G mutant enzymes. Thus, the KPC-3 variants examined here that cause resistance to ceftazidime-avibactam do not cause resistance to cefpodoxime-ETX1317.
Subject(s)
Azabicyclo Compounds , beta-Lactamases , Azabicyclo Compounds/pharmacology , Ceftazidime , Ceftizoxime/analogs & derivatives , Drug Combinations , Mutation , beta-Lactamases/genetics , CefpodoximeABSTRACT
ETX0282 is an orally bioavailable prodrug of the diazabicyclooctane serine ß-lactamase inhibitor ETX1317. The combination of ETX0282 with cefpodoxime proxetil is in clinical trials as an oral therapy for complicated urinary tract infections caused by Enterobacterales. Earlier diazabicyclooctane ß-lactamase inhibitors, such as avibactam and durlobactam, contain a sulfate moiety as the essential anionic group and are administered intravenously. In contrast, ETX1317 contains a fluoroacetate moiety, which is esterified with an isopropyl group in ETX0282 to provide high oral bioavailability. Previous studies of avibactam and durlobactam showed that covalent inhibition of certain ß-lactamases is reversible due to the ability of the ring-opened inhibitors to recyclize and dissociate in their original form. We investigated the interaction of ETX1317 with several ß-lactamases commonly found in relevant bacterial pathogens, including CTX-M-15, KPC-2, SHV-5, and TEM-1 from Ambler Class A; Pseudomonas aeruginosa AmpC and Enterobacter cloacae P99 from Class C, and OXA-48 from Class D. The second-order rate constants for inhibition (kinact/Ki) of these enzymes show that ETX1317 is intermediate in potency between durlobactam and avibactam. The partition ratios were all approximately 1, indicating that the inhibitor is not also a substrate of these enzymes. The rate constants for dissociation of the covalent complex (koff) were similar to those for durlobactam and avibactam. Acylation exchange experiments demonstrated that ETX1317 dissociated in its original form. No loss of mass from the inhibitor was observed in the covalent inhibitor-enzyme complexes.
Subject(s)
Serine , beta-Lactamase Inhibitors , Anti-Bacterial Agents/pharmacology , Enterobacter cloacae , beta-Lactamase Inhibitors/pharmacology , beta-LactamasesABSTRACT
The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, Burkholderia mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen ß-lactamase, which confers resistance to ß-lactams. The ß-lactam-ß-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. In this study, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured MICs of SUL-DUR against BCC and B. gladioli (n = 150), B. mallei (n = 30), and B. pseudomallei (n = 28), studied the kinetics of inhibition of the PenA1 ß-lactamase from B. multivorans and the PenI ß-lactamase from B. pseudomallei by durlobactam, tested for blaPenA1 induction by SUL-DUR, and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 µg/ml. Durlobactam potently inhibited PenA1 and PenI with second-order rate constant for inactivation (k2/K) values of 3.9 × 106 M-1 s-1 and 2.6 × 103 M-1 s-1 and apparent Ki (Kiapp) of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest that SUL-DUR may be useful as a treatment for Burkholderia infections.
Subject(s)
Burkholderia mallei , Burkholderia pseudomallei , Burkholderia , Glanders , Melioidosis , Animals , Anti-Bacterial Agents/pharmacology , Glanders/drug therapy , Horses , Melioidosis/drug therapy , Mice , Sulbactam/pharmacologyABSTRACT
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.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemistry , Formamides/chemistry , Hemodynamics/drug effects , Amidohydrolases/metabolism , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/therapeutic use , Binding Sites , Crystallography, X-Ray , Disease Models, Animal , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Escherichia coli/drug effects , Escherichia coli Infections/drug therapy , Escherichia coli Infections/pathology , Female , Formamides/metabolism , Formamides/pharmacology , Formamides/therapeutic use , Half-Life , Male , Mice , Molecular Dynamics Simulation , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Multidrug resistant Gram-negative bacterial infections are an increasing public health threat due to rapidly rising resistance toward ß-lactam antibiotics. The hydrolytic enzymes called ß-lactamases are responsible for a large proportion of the resistance phenotype. ß-Lactamase inhibitors (BLIs) can be administered in combination with ß-lactam antibiotics to negate the action of the ß-lactamases, thereby restoring activity of the ß-lactam. Newly developed BLIs offer some advantage over older BLIs in terms of enzymatic spectrum but are limited to the intravenous route of administration. Reported here is a novel, orally bioavailable diazabicyclooctane (DBO) ß-lactamase inhibitor. This new DBO, ETX1317, contains an endocyclic carbon-carbon double bond and a fluoroacetate activating group and exhibits broad spectrum activity against class A, C, and D serine ß-lactamases. The ester prodrug of ETX1317, ETX0282, is orally bioavailable and, in combination with cefpodoxime proxetil, is currently in development as an oral therapy for multidrug resistant and carbapenem-resistant Enterobacterales infections.
Subject(s)
Anti-Bacterial Agents/chemistry , Azabicyclo Compounds/chemistry , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/chemistry , Administration, Oral , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Azabicyclo Compounds/metabolism , Azabicyclo Compounds/pharmacology , Azabicyclo Compounds/therapeutic use , Drug Design , Drug Evaluation, Preclinical , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Half-Life , Humans , Mice , Microbial Sensitivity Tests , Penicillin-Binding Proteins/chemistry , Penicillin-Binding Proteins/metabolism , Prodrugs/chemistry , Prodrugs/metabolism , Protein Binding , Rats , Skin Diseases/drug therapy , Skin Diseases/pathology , Skin Diseases/veterinary , Structure-Activity Relationship , beta-Lactamase Inhibitors/metabolism , beta-Lactamase Inhibitors/pharmacology , beta-Lactamase Inhibitors/therapeutic use , beta-Lactamases/metabolismABSTRACT
Multi-drug-resistant Enterobacteriales expressing a wide array of ß-lactamases are emerging as a global health threat in both hospitals and communities. Although several intravenous drugs have recently been approved to address this need, there are no oral Gram-negative agents that are both safe and broadly effective against such pathogens. The lack of an effective oral agent is of concern for common infections which could otherwise be treated in the community but, due to antibiotic resistance, require hospitalization to allow for intravenous therapy. ETX1317 is a novel, broad spectrum, serine ß-lactamase inhibitor of the diazabicyclooctane class that restores the antibacterial activity of multiple ß-lactams against multiple species of multi-drug-resistant Enterobacteriales, including carbapenem-resistant strains. A combination of its oral prodrug, ETX0282, and the oral prodrug of a third-generation cephalosporin, cefpodoxime proxetil, is currently in clinical development. This report describes the biochemical and microbiological properties of ETX1317, which is more potent and demonstrates a greater breadth of inhibition than avibactam, the parenteral prototype of this class of ß-lactamase inhibitors.
Subject(s)
Pharmaceutical Preparations , beta-Lactamase Inhibitors , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Carbapenems , beta-Lactamase Inhibitors/pharmacology , beta-LactamsABSTRACT
The high-molecular mass penicillin-binding proteins (PBPs) are the essential targets of the ß-lactam classes of antibacterial drugs. In the Gram-negative pathogen Escherichia coli, these include PBP1a, PBP1b, PBP2, and PBP3. Techniques that enable facile measurement of the potency of inhibition of these targets are valuable for understanding structure-activity relationships in programs aimed at discovering new antibiotics to combat drug-resistant infections. Continuous fluorescence anisotropy-based assays for inhibition of soluble constructs of PBP1a, PBP2, and PBP3 from the serious Gram-negative bacterial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii and PBP3 from E. coli using the fluorescent phenoxypenicillin analogue BOCILLIN FL have been described previously, but this technique was not useful for PBP2 from E. coli due to a lack of change in fluorescence anisotropy or intensity upon reaction. Here, we report that a fluorescent analogue of ampicillin, 5-carboxytetramethylrhodamine-ampicillin (5-TAMRA-ampicillin), was useful as the indicator in a continuous fluorescence anisotropy-based kinetic assay for inhibition of a soluble construct of PBP2 from E. coli. The assay enables measurement of the bimolecular rate constant for inhibition kinact /Ki. This measurement was made for representative drugs from four classes of ß-lactams and for the diazabicyclooctenone ETX2514. 5-TAMRA-ampicillin was also useful in a fluorescence anisotropy-based assay for P. aeruginosa PBP2 and in fluorescence intensity-based assays with PBP1a and PBP3 from P. aeruginosa and A. baumannii and PBP3 from E. coli.
Subject(s)
Ampicillin/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli/drug effects , Penicillin-Binding Proteins/antagonists & inhibitors , Peptidyl Transferases/antagonists & inhibitors , Rhodamines/pharmacology , Acinetobacter baumannii/enzymology , Ampicillin/analogs & derivatives , Anti-Bacterial Agents/pharmacology , Escherichia coli/enzymology , Fluorescence Polarization , Kinetics , Microbial Sensitivity Tests , Pseudomonas aeruginosa/enzymology , beta-Lactams/pharmacologyABSTRACT
The novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine ß-lactamase inhibitor that restores sulbactam activity against resistant Acinetobacter baumannii The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10-10 to <9.0 × 10-10 at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to ß-lactamase inhibition, ETX2514 may enhance sulbactam activity in A. baumannii via inhibition of PBP2.
Subject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Gene Expression Regulation, Bacterial , Sulbactam/pharmacology , beta-Lactamase Inhibitors/pharmacology , Acinetobacter Infections/microbiology , Acinetobacter baumannii/genetics , Acinetobacter baumannii/isolation & purification , Acinetobacter baumannii/metabolism , Binding Sites , Drug Resistance, Multiple, Bacterial/genetics , Drug Therapy, Combination , Humans , Microbial Sensitivity Tests , Models, Molecular , Mutation , Penicillin-Binding Proteins/antagonists & inhibitors , Penicillin-Binding Proteins/chemistry , Penicillin-Binding Proteins/genetics , Penicillin-Binding Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
Sulbactam is one of four ß-lactamase inhibitors in current clinical use to counteract drug resistance caused by degradation of ß-lactam antibiotics by these bacterial enzymes. As a ß-lactam itself, sulbactam is susceptible to degradation by ß-lactamases. I investigated the Michaelis-Menten kinetics of sulbactam hydrolysis by 14 ß-lactamases, representing clinically widespread groups within all four Ambler classes, i.e., CTX-M-15, KPC-2, SHV-5, and TEM-1 for class A; IMP-1, NDM-1, and VIM-1 for class B; Acinetobacter baumannii ADC-7, Pseudomonas aeruginosa AmpC, and Enterobacter cloacae P99 for class C; and OXA-10, OXA-23, OXA-24, and OXA-48 for class D. All of the ß-lactamases were able to hydrolyze sulbactam, although they varied widely in their kinetic constants for the reaction, even within each class. I also investigated the inactivation kinetics of the inhibition of these enzymes by sulbactam. The class A ß-lactamases varied widely in their susceptibility to inhibition, the class C and D enzymes were very weakly inhibited, and the class B enzymes were essentially or completely unaffected. In addition, we measured the sulbactam turnover number, the sulbactam/enzyme molar ratio required for complete inhibition of each enzyme. Class C enzymes had the lowest turnover numbers, class A enzymes varied widely, and class D enzymes had very high turnover numbers. These results are valuable for understanding which ß-lactamases ought to be well inhibited by sulbactam. Moreover, since sulbactam has intrinsic antibacterial activity against Acinetobacter species pathogens, these results contribute to understanding ß-lactamase-mediated sulbactam resistance in Acinetobacter, especially due to the action of the widespread class D enzymes.
Subject(s)
Acinetobacter baumannii/chemistry , Enterobacter cloacae/chemistry , Pseudomonas aeruginosa/chemistry , Sulbactam/metabolism , beta-Lactamase Inhibitors/metabolism , beta-Lactamases/metabolism , Acinetobacter baumannii/enzymology , Acinetobacter baumannii/genetics , Enterobacter cloacae/enzymology , Enterobacter cloacae/genetics , Gene Expression , Hydrolysis , Kinetics , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Species Specificity , Sulbactam/pharmacology , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/classification , beta-Lactamases/genetics , beta-Lactamases/isolation & purificationABSTRACT
ETX2514 is a non-ß-lactam serine ß-lactamase inhibitor in clinical development that has greater potency and broader spectrum of ß-lactamase inhibition than the related diazabicyclooctanone avibactam. Despite opening of its cyclic urea ring upon acylation, avibactam can recyclize and dissociate intact from certain ß-lactamases. We investigated reversibility of ETX2514 acylation of 10 serine ß-lactamases representing Ambler classes A, C, and D. Dissociation rate constants varied widely between enzymes and were lowest for class D. For most enzymes, the covalent adduct mass was that of ETX2514 (277 Da). OXA-10 was acylated with 277 and 197 Da adducts, consistent with loss of the sulfate moiety. KPC-2 showed only the 197 Da adduct. ETX2514 recyclized and dissociated intact from AmpC, CTX-M-15, P99, SHV-5 and TEM-1 but not from KPC-2, OXA-10, OXA-23, OXA-24, or OXA-48. Inactivation partition ratios were 1 for all enzymes except KPC-2, for which it increased to 3.0 after 2 h. This result and mass spectrometry showed that KPC-2 very slowly degraded ETX2514. Nevertheless, ETX2514 restored ß-lactam activity to equal potency against isogenic Pseudomonas aeruginosa strains each overexpressing one of the 10 ß-lactamases.
Subject(s)
Pseudomonas aeruginosa/genetics , Sulfones/chemistry , beta-Lactamase Inhibitors/pharmacology , Anti-Bacterial Agents/chemistry , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/pharmacology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/metabolismABSTRACT
Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of ß-lactamases, enzymes that inactivate ß-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new ß-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine ß-lactamase inhibitors that potently inhibit clinically relevant class A, C and D ß-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of ß-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.
Subject(s)
Acinetobacter baumannii/drug effects , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/pharmacology , Gram-Negative Bacteria/drug effects , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Animals , Azabicyclo Compounds/therapeutic use , Azabicyclo Compounds/toxicity , Carbapenems/pharmacology , Dogs , Drug Design , Drug Evaluation, Preclinical , Drug Resistance, Multiple, Bacterial , Enterobacteriaceae/drug effects , Gram-Negative Bacterial Infections/drug therapy , Humans , Mice , Models, Molecular , Penicillin-Binding Proteins/antagonists & inhibitors , Rats , Sulbactam/chemistry , Sulbactam/pharmacology , beta-Lactamase Inhibitors/therapeutic use , beta-Lactamase Inhibitors/toxicity , beta-Lactamases/metabolism , beta-Lactams/pharmacologyABSTRACT
Fatty acid biosynthesis is essential to bacterial growth in Gram-negative pathogens. Several small molecules identified through a combination of high-throughput and fragment screening were cocrystallized with FabH (ß-ketoacyl-acyl carrier protein synthase III) from Escherichia coli and Streptococcus pneumoniae. Structure-based drug design was used to merge several scaffolds to provide a new class of inhibitors. After optimization for Gram-negative enzyme inhibitory potency, several compounds demonstrated antimicrobial activity against an efflux-negative strain of Haemophilus influenzae. Mutants resistant to these compounds had mutations in the FabH gene near the catalytic triad, validating FabH as a target for antimicrobial drug discovery.
Subject(s)
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Drug Resistance, Bacterial , Enzyme Inhibitors/pharmacology , Haemophilus influenzae/enzymology , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/chemistry , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/metabolism , Anti-Bacterial Agents/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Design , Enzyme Inhibitors/chemistry , Haemophilus Infections/microbiology , Haemophilus influenzae/chemistry , Haemophilus influenzae/drug effects , Haemophilus influenzae/genetics , Humans , Microbial Sensitivity Tests , MutationABSTRACT
This review covers the uses of fluorescence polarization and anisotropy for the investigation of bacterial penicillin binding proteins (PBPs), which are the targets of ß-lactam antibacterial drugs (penicillins, cephalosporins, carbapenems, and monobactams), and of the ß-lactamase enzymes that destroy these drugs and help to render bacterial pathogens resistant to them. Fluorescence polarization and anisotropy-based methods for quantitation of ß-lactam drugs are also reviewed. A particular emphasis is on methods for quantitative measurement of the interactions of ß-lactams and other inhibitors with PBPs and ß-lactamases.
Subject(s)
Anisotropy , Anti-Bacterial Agents , Fluorescence Polarization , Penicillin-Binding Proteins , beta-Lactamases , beta-LactamsABSTRACT
We present the combination of the clinically well-proven chemotherapeutic agent, Doxorubicin, and (99m)Tc, an Auger and internal conversion electron emitter, into a dual-action agent for therapy. Chemical conjugation of Doxorubicin to (99m)Tc afforded a construct which autonomously ferries a radioactive payload into the cell nucleus. At this site, damage is exerted by dose deposition from Auger radiation. The (99m)Tc-conjugate exhibited a dose-dependent inhibition of survival in a selected panel of cancer cells and an in vivo study in healthy mice evidenced a biodistribution which is comparable to that of the parent drug. The homologous Rhenium conjugate was found to effectively bind to DNA, inhibited human Topoisomerase II, and exhibited cytotoxicity in vitro. The collective in vitro and in vivo data demonstrate that the presented metallo-conjugates closely mimic native Doxorubicin.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Doxorubicin/chemistry , Doxorubicin/pharmacology , Technetium/chemistry , Technetium/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Survival/drug effects , DNA Topoisomerases, Type II/metabolism , Doxorubicin/pharmacokinetics , Humans , Mice , Neoplasms/drug therapy , Technetium/pharmacokinetics , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacokinetics , Topoisomerase II Inhibitors/pharmacologyABSTRACT
We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and ß.