In Vitro and in Vivo Evaluation of Metal- Chelating Agents as Novel Metallo Beta-Lactamase Inhibitors Against Carbapenem -Resistant Enterobacteriaceae. 2018 (original) (raw)
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In vitro evaluation of metal chelators as potential metallo- β -lactamase inhibitors
Journal of Applied Microbiology, 2016
Aims: This study aimed at investigating the use of metal chelators as potential metallo-b-lactamase inhibitors (MBL). Methods and Results: The minimum inhibitory concentration (MIC) of meropenem was ascertained alone and in combination with various concentrations of macrocyclic (1,4,7-triazacyclononane-1-glutaric acid-4,7diacetic acid = NODAGA) peptide derivatives and acyclic (N,N,N 0 ,N 0-Tetrakis(2pyridylmethyl)ethylenediamine = TPEN and di-(2-picolyl)amine = DPA) metal chelators using the broth microdilution method. MICs of meropenem against carbapenem-resistant enterobacteriaceae (CRE) producing MBLs were decreased to concentrations as low as 0Á06 mg l À1 in the presence of some metal chelators. TPEN at 4 and 8 mg l À1 showed the best activity by decreasing meropenem MICs to 0Á5 and 0Á06 mg l À1 , respectively, for some New Delhi Metallo-beta-lactamase (NDM) and Verona integron-encoded metallo-b-lactamase (VIM)-producing enterobacteriaceae. DPA at 8 and 16 mg l À1 was also able to decrease meropenem MICs to 1 and 0Á125 mg l À1 , respectively, for these CREs. NODAGA peptide derivatives showed the least inhibition as 32 mg l À1 was required for meropenem MICs to be decreased to 0Á06 mg l À1 against an NDM-1 producing isolate. Conclusion: The various metal chelators, TPEN, DPA and NODAGA peptide derivatives were able to inhibit the MBLs in decreasing order of activity, rendering CREs susceptible to meropenem. Significance and Impact of the Study: In the absence of new antibiotics, this study evaluated metal chelators as potential MBL inhibitors.
bioRxiv (Cold Spring Harbor Laboratory), 2018
Metallo--lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo--lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available -lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae. MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACNmeropenem combination showed bactericidal effects with an MBC/MIC ratio of Յ4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic activesite pockets. This was supported by its inhibition constant (K i), which was 0.044 M, and its inactivation constant (K inact), which was 0.0406 min Ϫ1 , demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor. IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo--lactamases (MBLs), which inactivate -lactam antibiotics. Combination therapies involving a -lactam antibiotic and a -lactamase inhibitor remain a major treatment option for infections caused by -lactamase-producing organisms. Currently, no MBL inhibitor--lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.
ACS Infectious Diseases
In the search for new inhibitors of bacterial metalloβ-lactamases (MBLs), a series of commonly used small molecule carboxylic acid derivatives were evaluated for their ability to inhibit New Delhi metallo-β-lactamase (NDM)-, Verona integronencoded metallo-β-lactamase (VIM)-, and imipenemase (IMP)type enzymes. Nitrilotriacetic acid (3) and N-(phosphonomethyl)iminodiacetic acid (5) showed promising activity especially against NDM-1 and VIM-2 with IC 50 values in the low-to-sub μM range. Binding assays using isothermal titration calorimetry reveal that 3 and 5 bind zinc with high affinity with dissociation constant (K d) values of 121 and 56 nM, respectively. The in vitro biological activity of 3 and 5 against E. coli expressing NDM-1 was evaluated in checkerboard format, demonstrating a strong synergistic relationship for both compounds when combined with Meropenem. Compounds 3 and 5 were then tested against 35 pathogenic strains expressing MBLs of the NDM, VIM, or IMP classes. Notably, when combined with Meropenem, compounds 3 and 5 were found to lower the minimum inhibitory concentration (MIC) of Meropenem up to 128-fold against strains producing NDM-and VIM-type enzymes.
The clinical effectiveness of the important β-lactam class of antibiotics is under threat by the emergence of resistance, mostly due to the production of acquired serine-(SBL) and metalloβ-lactamase (MBL) enzymes. To address this resistance issue, multiple β-lactam/ β-lactamase inhibitor combinations have been successfully introduced into the clinic over the past several decades. However, all of those combinations contain SBL inhibitors and, as yet, there are no MBL inhibitors in clinical use. Consequently, there exists an unaddressed yet growing healthcare problem due to the rise in recent years of highly resistant strains which produce New Delhi Metallo (NDM)-type metallo-carbapenemases. Previously we reported the characterisation of an advanced MBL inhibitor lead compound, ANT431. Herein we discuss the completion of a lead optimisation campaign culminating in the discovery of the preclinical candidate ANT2681, a potent NDM inhibitor with strong potential for clinical development.
Antibiotics
Virulent Enterobacterale strains expressing serine and metallo-β-lactamases (MBL) genes have emerged responsible for conferring resistance to hard-to-treat infectious diseases. One strategy that exists is to develop β-lactamase inhibitors to counter this resistance. Currently, serine β-lactamase inhibitors (SBLIs) are in therapeutic use. However, an urgent global need for clinical metallo-β-lactamase inhibitors (MBLIs) has become dire. To address this problem, this study evaluated BP2, a novel beta-lactam-derived β-lactamase inhibitor, co-administered with meropenem. According to the antimicrobial susceptibility results, BP2 potentiates the synergistic activity of meropenem to a minimum inhibitory concentration (MIC) of ≤1 mg/L. In addition, BP2 is bactericidal over 24 h and safe to administer at the selected concentrations. Enzyme inhibition kinetics showed that BP2 had an apparent inhibitory constant (Kiapp) of 35.3 µM and 30.9 µM against New Delhi Metallo-β-lactamase (NDM-1) and ...
Microbial Drug Resistance
Infections caused by metallo-b-lactamase (MBL)-producing bacteria are emerging and carry a significant impact on patients' outcome. MBL producers are spread worldwide, both in community and hospital setting, with increasingly reported epidemic clusters and the search for MBL inhibitors is an important topic for public health. MBLs are zinc-dependent enzymes whose functioning can be hampered by zinc chelators. We evaluated the potential of six zinc chelators (disulfiram, nitroxoline, 5-amino-8-hydroxyquinoline, 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA], cyclam, and N,N,N¢,N¢-tetrakis (2-pyridymethyl) ethylenediamine [TPEN]) in restoring carbapenem activity against MBL producers. Zinc chelators alone or in combination with meropenem against MBL-producing Klebsiella pneumoniae, Chryseobacterium indologenes, Elizabethkingia meningoseptica, and Stenotrophomonas maltophilia isolates were tested in vitro and in vivo (Galleria mellonella). In vitro experiments showed a synergistic activity between TPEN and meropenem toward all the strains. Nitroxoline alone retained activity against S. maltophilia, C. indologenes, and E. meningoseptica. In vivo experiments showed that TPEN or nitroxoline in combination with meropenem increased survival in larvae infected with E. meningoseptica, S. maltophilia, and K. pneumoniae. Based on our data, zinc chelators are potential carbapenem adjuvants molecules (restoring carbapenem activity) against MBL-sustained infections and could represent an interesting option for infections induced by these microorganisms.
Metallo-β-lactamases in Gram-negative bacteria: introducing the era of pan-resistance?
International Journal of Antimicrobial Agents, 2009
Metallo--lactamases (MBLs) are being reported with increasing frequency and from several countries worldwide and are becoming the prevalent and most clinically significant determinants of carbapenem resistance. Furthermore, MBL-producing strains that exhibit a pan-resistant phenotype are increasingly detected. Initially MBLs were detected in Pseudomonas aeruginosa, however nowadays they are frequently found in Acinetobacter baumannii, Klebsiella pneumoniae and other Enterobacteriaceae. MBLs spread easily on plasmids and cause nosocomial infections and outbreaks with excess mortality. Such infections mainly concern patients admitted to Intensive Care Units with several co-morbidities and a history of prolonged administration of antibiotics. MBL-producing strains exhibit resistance to almost all currently available antibiotics. In vitro studies reveal that tigecycline and colistin are the only antibacterial agents with consistent activity against MBL-producing strains. Randomised controlled trials are required in order to evaluate the available therapeutic regimens, including treatment combinations. Tigecycline and colistin should be used under appropriate prescribing practices. Surveillance to monitor the emergence of resistance to these agents as well as implementation of infection control measures should be strengthened. MBL inhibitors are urgently needed, however, none is in late pre-clinical development.
Applied and Environmental Microbiology, 2018
Metallo-β-lactamase producing Enterobacteriaceae are of grave clinical concern particularly as there are no Metallo-β-lactamase (MBL) inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1, 4, 7-triazacyclononane (TACN) for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), serum effect, fractional inhibitory concentrations index and time-kill kinetics were performed using broth microdilution techniques according to the Clinical Laboratory Standard Institute (CSLI) guidelines. Enzyme kinetic parameters and cytotoxicity effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-produc...
Synthesis and biological evaluation of novel β-lactam-metallo β-lactamase inhibitors
RSC Advances, 2023
b-lactamases are enzymes that deactivate b-lactam antibiotics through a hydrolysis mechanism. There are two known types of b-lactamases: serine b-lactamases (SBLs) and metallo b-lactamases (MBLs). The two existing strategies to overcome b-lactamase-mediated resistance are (a) to develop novel b-lactam antibiotics that are not susceptible to hydrolysis by these enzymes; or (b) to develop b-lactamase inhibitors that deactivate the enzyme and thereby restore the efficacy of the co-administered antibiotics. Many commercially available SBL inhibitors are used in combination therapy with antibiotics to treat antimicrobial resistant infections; however, there are only a handful of MBL inhibitors undergoing clinical trials. In this study, we present 11 novel potential MBL inhibitors (via multi-step chemical synthesis), that have shown to completely restore the efficacy of meropenem (#2 mg L −1) against New Delhi metallo-b-lactamase (NDM) producing Klebsiella pneumoniae in vitro. These compounds contain a cyclic amino acid zinc chelator conjugated to various commercially available b-lactam antibiotic scaffolds with the aim to improve the overall drug transport, lipophilicity, and pharmacokinetic/pharmacodynamic properties as compared to the chelator alone. Biological evaluation of compounds 24b and 24c has further highlighted the downstream application of these MBLs, since they are non-toxic at the selected doses. Time-kill assays indicate that compounds 24b and 24c exhibit sterilizing activity towards NDM producing Klebsiella pneumoniae in vitro using minimal concentrations of meropenem. Furthermore, 24b and 24c proved to be promising inhibitors of VIM-2 (K i = 0.85 and 1.87, respectively). This study has revealed a novel series of b-lactam MBLIs that are potent, efficacious, and safe leads with the potential to develop into therapeutic MBLIs. cantly to antibiotic resistance. 1,3,4 The Antimicrobial Resistance (AMR) National Strategy Framework (2018-2024), as well as the Global Research and Development priority setting for AMR, and the Global Antibiotic Research and Development Partnership, prioritise research regarding antimicrobial treatment to help prevent obsolete antibiotics emanating as a result of mutations and bacterial evolution. 5-7 Antibiotic resistance has worsened, due to the empirical treatment of hospitalised COVID-19 patients. 3,8,9 There are several initiatives in place to either raise awareness, help reduce, or keep track of resistance, such as: the Global Action Plan on Antimicrobial Resistance (GAP); World Antimicrobial Awareness Week (WAAW); The Global Antimicrobial Resistance and Use Surveillance System (GLASS); Global Research and Development priority setting for AMR; Access Watch Reserve (AWaRe); and Global Antibiotic Research and Development Partnership (GARDP). 7 Murray et al. estimated that 4.95 million deaths occurred in the year 2019 as a result of antibiotic resistance. 6 Identied Klebsiella pneumoniae, as a common pathogen accounting for 29% of all reported bacterial infections. 7 Klebsiella pneumoniae has recently been identied as a bacteria of concern, as mentioned in several reports, studies, and reviews, and is further substantiated by the resistance map (Fig. 1) generated from the CDDEP (Centre for Disease Dynamics, Economics and Policy). 10 Unfortunately,