In vitro and computational studies of the ß-lactamase inhibition and ß-lactam potentiating properties of plant secondary metabolites.

ß-lactam resistance in bacteria is primarily mediated through the production of ß-lactamases. Among the several strategies explored to mitigate the issue of ß-lactam resistance, the use of plant secondary metabolites in combination with existing ß-lactams seem promising. The present study aims to identify possible ß-lactam potentiating plant secondary metabolites following in vitro and in silico approaches. Among 180 extracts from selected 30 medicinal plants, acetone extract of Ficus religiosa (FRAE) bark recorded the least IC50 value of 3.9 mg/ml. Under in vitro conditions, FRAE potentiated the activity of ampicillin, which was evidenced by the significant reduction in IC50 values of ampicillin against multidrug resistant bacteria. Metabolic profiling following HR-LCMS analysis revealed the presence of diverse metabolites viz. flavonoids, alkaloids, terpenoids, etc. in FRAE. Further, ensemble docking of the FRAE metabolites against four Class A ß-lactamase (SHV1, TEM1, KPC2 and CTX-M-27) showed quercetin, taxifolin, myricetin, luteolin, and miquelianin as potential inhibitors with the least average binding energy. In molecular dynamic simulation studies, myricetin formed the most stable complex with SHV1 and KPC-2 while miquelianin with TEM1 and CTX-M-27. Further, all five metabolites interacted with amino acid residue Glu166 in Ω loop of ß-lactamase, interfering with the deacylation step, thereby disrupting the enzyme activity. The pharmacokinetics and ADMET profile indicate their drug-likeness and non-toxic nature, making them ideal ß-lactam potentiators. This study highlights the ability of metabolites present in FRAE to act as ß-lactamase inhibitors.Communicated by Ramaswamy H. Sarma.

Synergistic evaluation of Moringa oleifera extract and ß-lactam antibiotic to restore the susceptibility of methicillin-resistant Staphylococcus aureus.

INTRODUCTION: The antibiotic resistance has become a major threat to global health. The combinatorial use of two or more compounds to develop a new formulation may overcome the emerging cases of drug resistance. Moringa oleifera has been utilized as a strong nutritional, immunomodulator and therapeutic agent for decades. In this study, different parts of Moringa oleifera were screened for bioactive compounds that can act as a resistance modifying agent for multi-drug resistant organisms (MDROs). METHODOLOGY: Initially, the combined effect of stem bark extracts and ampicillin was calculated by checkerboard assay. Active compounds of effective extract were assessed by High Performance Liquid Chromatography (HPLC). Minimal Inhibitory Concentration (MIC) and Fractional Inhibitory Concentration Index (FICI) were calculated to evaluate the synergistic behavior of stem bark extract with ampicillin. To study the blocking of resistance pathways of Methicillin-Resistant Staphylococcus aureus (MRSA) western blot was performed. RESULTS: The results revealed that stem bark has significant anti-MRSA activity. The methanolic extract of stem bark in combination with ampicillin showed the highest synergistic effect (FICI value ≤ 0.237) against MRSA. Killing kinetics and membrane potential of ampicillin alone and in combination revealed an increase in the inhibitory potential of ampicillin against MRSA. Decolourization in iodometric assay confirmed the inhibition of ß-lactamase, western blot results confirmed the blocking of penicillin-binding protein (PBP2a) expression with the restoration of MRSA sensitivity against ß-lactams. CONCLUSION: It can be concluded that methanolic extract of Moringa oleifera stem bark has bioactive compounds and can be used as an adjuvant with antibiotics to modify the resistance of MDROs.

The potentiation of beta-lactam and anti-bacterial activities of lipophilic constituents from Mesua ferrae leaves against methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Mesua ferrae, from the family of Calophyllaceae, is traditionally used for the treatment of piles, fever and renal disorders. The present study was aimed to examine the antibacterial compounds from the leaves of M. ferrae and their ß-lactam antibiotic potentiate activities against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Stigmasterol (1) and ß-caryophyllene oxide (2) were isolated from the n-hexane fraction of the leaves of M. ferrae using a bioassay-guided fractionation approach. RESULTS: The isolated compounds displayed anti-Staphylococcus and anti-MRSA activities. It is worth to note that both compounds demonstrated synergism with ß-lactam antibiotics against S. aureus and MRSA. Gas chromatography-mass spectrometry (GC-MS) analysis indicated the n-hexane fraction was dominated by triterpenes and sesquiterpenes, suggesting the total antibacterial activity exhibited by the fraction. CONCLUSION: Based on the findings, it could conclude that M. ferrae is a promising natural source for the discovery of new anti-MRSA lead compounds.

Polypharmacological drug actions of recently FDA approved antibiotics.

The current epidemic of antibiotic resistant bacterial infections has fueled the demand for novel antibiotics exhibiting both antibacterial efficacy and anti-drug resistance. This need has not been fully satisfied by the conventional "one target-one molecule" approach. Consequently, there has been rising interest in the development of multi-target antibiotics. Over the past two decades, 52% (14 out of 27) of the FDA approved antibiotics have demonstrated synergistic, multi-target mechanisms of action. Among these are three second-generation lipoglycopeptides, five new generation quinolones and six modernized ß-lactams. This review focuses on the structure-activity relationship (SAR) analysis and the polypharmacological drug action of these antibiotics, to reveal how these multi-target antibiotics achieve the dual objectives of maximizing bactericidal or bacteriostatic efficacy and minimizing antibiotic resistance. The entrance of multi-target antibiotics into the FDA-approved regimens represents a milestone in the evolution of drug discovery as it has transcended from chemical library screening to rational drug design.

Harnessing ß-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis.

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 ß-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b, or PBP4. Almost all penicillin scaffolds were coselective for all or combinations of PBP2a, 2b, and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin) and 2b (cefotaxime) or 2a, 2b, and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime, and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid), and various coselective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for ß-lactams and speculated to be involved in ß-lactam resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

Three-component synthesis of chromeno ß-lactam hybrids for inflammation and cancer screening.

Highly diastereoselective synthesis of chromeno ß-lactam hybrids was achieved by an efficient one-pot three-component reaction. With this procedure, the desired ß-lactam products were obtained in good yields and with exclusive cis stereoselection, by combining a variety of benzaldehydes, malononitrile, and either 5,5-dimethylcyclohexane-1,3-dione or 4-hydroxycoumarin in the presence of 1,4-diazabicyclo [2.2.2]octane under reflux conditions. These adducts were structurally characterized on the basis of IR, 1D and 2D NMR spectra, X-ray analysis, H-H COSY and H-C HSQC two-dimensional NMR experiments, and elemental analysis. Each of the synthesized compounds was screened for anti-inflammatory and anticancer activities. ß-Lactams 5b and 8b showed a 53.4 and 19.8 anti-inflammatory ratio, respectively, and 5b appeared more active than the well-known dexamethasone corticosteroid used for the treatment of rheumatoid and skin inflammation. ß-Lactams 5a, 5b, 5e, 5f, 5g, 8c, 8j and 8p also showed good antitumor activity against the SW1116 (colon cancer) cell line without notable cytotoxicity towards the HepG2 control cell line.

Effect of non-ß-lactams on stable variants of inhibitor-resistant TEM ß-lactamase in uropathogenic Escherichia coli: implication for alternative therapy.

AIMS: ß-lactamase inhibitor resistance (BLIR) among the uropathogenic Escherichia coli (UPEC) minimizes treatment options. This study aimed to identify inhibitor-resistant TEM (IRT) ß-lactamase that impart BLIR phenotype and explore non-ß-lactams as alternative therapeutics. METHODS AND RESULTS: Thirty BLIR UPEC isolates were detected by Kirby-Bauer disc diffusion technique using ß-lactam-ß-lactamase inhibitor combination. Conjugal transfer of BLIR was successful from 17 isolates. PCR and sequencing of the TEM ß-lactamases from the transconjugants indicated 14 TEM-84 (IRT) and three novel IRT variants (pUE184TEM, pUE203TEM, pUE210TEM). Three-dimensional models of the latter were predicted and validated. Molecular docking of selected non-ß-lactams (morin, catechin, naringenin triacetate) with the variants using AutoDock 4.2 showed comparable docking scores with significant hydrogen bond and hydrophobic interactions. Molecular dynamics simulation study confirmed stability of the non-ß-lactams inside the catalytic pocket of the enzymes. Moreover, all three non-ß-lactams were found to inhibit the purified TEM ß-lactamase variants in vitro. Microbroth dilution method indicated naringenin triacetate 64 µg ml-1 in combination with ceftazidime (CAZ) 30 µg ml-1 to be most effective against the BLIR transconjugants. CONCLUSIONS: BLIR phenotypes were primarily attributed to the production of IRT ß-lactamases. Administration of the non-ß-lactams with CAZ demonstrated an alternative therapeutic strategy against the IRT ß-lactamase producers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates high risk of transmission of IRT ß-lactamases and suggests ß-lactam-non-ß-lactam combination therapy to combat BLIR.

Novel activity-based probes for N-acylethanolamine acid amidase.

The cysteine hydrolase, N-acylethanolamine acid amidase (NAAA) is a promising target for analgesic and anti-inflammatory drugs. Here, we describe the development of two unprecedented NAAA-reactive activity-based probes as research tools for application in the discovery of new inhibitors and for the in-depth characterization of NAAA in its cellular environment.

Synergistic effect of (+)-pinitol from Saraca asoca with ß-lactam antibiotics and studies on the in silico possible mechanism.

Saraca asoca bark has been used in the Ayurvedic system of medicine for female urino-genital disorders. We have recently reported the isolation and characterization of several compounds as markers to develop HPLC profiling of its methanol and aqueous methanol extracts. Now, a HPLC-PDA inactive compound, (+)-pinitol has been isolated and characterized from the bark of this medicinally important tree. Pinitol is a well known bioactive compound for a variety of biological activities, including hypoglycemic and anti-inflammatory activities. A process for the isolation of relatively good concentration of (+)-pinitol from S. asoca bark has been developed and its in vitro anti TNF-α and anti-inflammatory activities against carragenan-induced edema confirmed. While conducting experiments on the possible agonistic activity, it was found that (+)-pinitol showed up to eight fold reduction in the doses of ß-lactam antibiotics. The mechanism of its agonistic activity was studied by docking experiments which showed that different conformations of (+)-pinitol and antibiotics were actually in the same binding site with no significant change in the binding energy. These docking simulations, thus predict the possible binding mode of studied compounds and probable reason behind the synergistic effect of (+)-pinitol along with ß-lactam antibiotics.

Bioactivity Studies of ß-Lactam Derived Polycyclic Fused Pyrroli-Dine/Pyrrolizidine Derivatives in Dentistry: In Vitro, In Vivo and In Silico Studies.

The antibacterial activity of ß-lactam derived polycyclic fused pyrrolidine/pyrrolizidine derivatives synthesized by 1, 3-dipolar cycloaddition reaction was evaluated against microbes involved in dental infection. Fifteen compounds were screened; among them compound 3 showed efficient antibacterial activity in an ex vivo dentinal tubule model and in vivo mice infectious model. In silico docking studies showed greater affinity to penicillin binding protein. Cell damage was observed under Scanning Electron Microscopy (SEM) which was further proved by Confocal Laser Scanning Microscope (CLSM) and quantified using Flow Cytometry by PI up-take. Compound 3 treated E. faecalis showed ROS generation and loss of membrane integrity was quantified by flow cytometry. Compound 3 was also found to be active against resistant E. faecalis strains isolated from failed root canal treatment cases. Further, compound 3 was found to be hemocompatible, not cytotoxic to normal mammalian NIH 3T3 cells and non mutagenic. It was concluded that ß-lactam compound 3 exhibited promising antibacterial activity against E. faecalis involved in root canal infections and the mechanism of action was deciphered. The results of this research can be further implicated in the development of potent antibacterial medicaments with applications in dentistry.

A novel ß-lactam derivative, albactam from the flowers of Albizia lebbeck with platelets anti-aggregatory activity in vitro.

A novel ß-lactam derivative, albactam, was isolated from the alcoholic extract of the flowers of Albizia lebbeck. It showed a significant anti-aggregatory activity against adenosine diphosphate and arachidonic acid induced guinea-pigs' platelets aggregation in vitro. Six more known compounds were also isolated and fully characterized by measuring 1D and 2D NMR, two of them are the triterpenes ß-amyrin and 11α, 12α-oxidotaraxerol, two ceramide derivatives and two flavonoids, kampferol 3-O-rutinoside and rutin.

Fluorescent TEM-1 ß-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening.

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 ß-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when ß-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent ß-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type ß-lactamase variants for in vitro drug screening.

4-Aminoquinoline-ß-lactam conjugates: synthesis, antimalarial, and antitubercular evaluation.

A library of quinoline-ß-lactam-based hybrids was synthesized and tested for their antimalarial and antitubercular activities. The present antimalarial data showed the dependence of activity on the nature of linker, N-1 substituent of the ß-lactam ring as well as the length of alkyl chain. Most of the compounds are not as efficient as chloroquine in inhibiting the culture growth of Plasmodium falciparum W2 strain. Nevertheless, the synthesized hybrids showed better antitubercular activities (up to five times) compared with cephalexin (up to three times) and ethionamide.

Synthetic approach toward alpha-aminomethyl-gamma-butyrolactones from beta-lactam synthons elaborated by SmI2-mediated reductive coupling reactions.

A simple and straightforward synthetic approach was developed to access a biologically important class of alpha-aminomethyl-gamma-butyrolactones via a beta-lactam synthon strategy involving successive ring-opening and lactonization processes from alpha-hydroxyethyl-substituted beta-lactams that were elaborated by SmI2-mediated reductive coupling reaction.

Discovery of wall teichoic acid inhibitors as potential anti-MRSA ß-lactam combination agents.

Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore ß-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA ß-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.

Allenyl-ß-lactams: versatile scaffolds for the synthesis of heterocycles.

The hybrid allenic ß-lactam moiety represents an excellent building block for carbo- and heterocyclization reactions, affording a large number of cyclic structures containing different sized skeletons in a single step. This strategy has been studied under thermal and radical-induced conditions. More recently, the use of transition-metal catalysis has been introduced as an alternative that relies on the activation of the allenic component. On the other hand, the intramolecular version has attracted much attention as a strategy for the synthesis of bi- and tricyclic compounds in a regio- and stereoselective manner. This overview focuses on the most recently developed cyclizations on 2-azetidinone-tethered allenes along with remarkable early works accounting for the mechanism, as well as for the regio- and diastereoselectivities of the cyclizations.

Synthesis of selenium-containing bicyclic beta-lactams via alkene metathesis.

The stereoselective insertion of allyl-seleno moieties at the C(4) position of azetidinones and further ring-closing metathesis afforded novel selenium-containing bicyclic beta-lactams.

[Ertapenem and other antibacterials].

Ertapenem is a carbapenem that shares the activity of imipenem and meropenem against most species, but is less active against non-fermenters. The activity is retained against most strains with AmpC and extended-spectrum beta-lactamases, although the resistance can arise if these enzymes are combined with extreme impermeability. The resistance can also be caused by IMP, VIM, KPC and NMC carbapenemases, but again, co-requires impermeability. Although the spread of carbapenemases in the future is a concern, they are currently very rare. Given as a 1 g intravenous (i.v.) infusion once daily, ertapenem has a plasma half-life of -4 h in healthy volunteers, and a Cn,a, of 155 mg/l and 13 mg/l for total and free drug, respectively. Excretion is largely renal, divided equally between native drug and an open-ring derivative. The trials showed equivalence to piperacillin/tazobactam or ceftriaxone in (a) abdominal infections, (b) community-acquired pneumonia, (c) acute pelvic infections, (d) skin and skin structure infections and (e) complicated urinary tract infections. The USA licence grants all these five indications; the ED licence grants the first three. Further potential uses include home i.v. therapy, target therapy against Enterobacteriaceae with AmpC or extended-spectrum cephalosporinases, and tentatively, surgical prophylaxis. Widening the usage of carbapenems raises public health concerns, somewhat allayed by the continued rarity of carbapenemases after 17 years of imipenem use, and by the fact that carbapenemases occur mostly in non-fermenters outside the spectrum of ertapenem, and co-require impermeability to confer resistance in Enterobacteriaceae. Nevertheless, if ertapenem is to be used widely, its effects on the microbial resistance ecology need to be monitored carefully.

Redefining penems.

The antimicrobial class of penems has the potential to address most of the relevant resistance issues associated with beta-lactam antibiotics because of their exceptionally broad spectrum of antibacterial activity and their intrinsic stability against hydrolytic attack by many beta-lactamases including ESBL and AmpC enzymes. The subclass of carbapenems covers the spectrum of hospital pathogens whereas the subclass of penems covers community pathogens. The only currently available penem, faropenem, has a low propensity for resistance development, beta-lactamase induction and selection of carbapenem-resistant Pseudomonas aeruginosa. This makes it attractive for the treatment of community-acquired infections and for step-down or sequential therapy following carbapenem treatment without jeopardizing the activity of carbapenems or the entire beta-lactam class in the hospital environment.

Asymmetric synthesis of beta-lactams through the Staudinger reaction and their use as building blocks of natural and nonnatural products.

In the last two decades, the better understanding of the mechanistic aspects of the beta-lactams' biological activity and their inhibition, and the chemical exploitation of beta-lactams as synthetic intermediates in organic chemistry, have experienced a continuous and somewhat complementary advance. A prerequisite for such a development has been the accessibility of enantiopure beta-lactams. The latter are now routinely prepared most often through the ketene-imine cycloaddition reaction, also termed the Staudinger reaction. This review accounts for the recent progress made in the asymmetric synthesis of beta-lactams (with special emphasis in the Staudinger reaction approach), as well as in their use as synthetic intermediates en route to natural products, including alpha- and beta-amino acids and peptides derived therefrom.