Discovery of YFJ-36: Design, Synthesis, and Antibacterial Activities of Catechol-Conjugated ß-Lactams against Gram-Negative Bacteria.

Cefiderocol is the first approved catechol-conjugated cephalosporin against multidrug-resistant Gram-negative bacteria, while its application was limited by poor chemical stability associated with the pyrrolidinium linker, moderate potency against Klebsiella pneumoniae and Acinetobacter baumannii, intricate procedures for salt preparation, and potential hypersensitivity. To address these issues, a series of novel catechol-conjugated derivatives were designed, synthesized, and evaluated. Extensive structure-activity relationships and structure-metabolism relationships (SMR) were conducted, leading to the discovery of a promising compound 86b (Code no. YFJ-36) with a new thioether linker. 86b exhibited superior and broad-spectrum in vitro antibacterial activity, especially against A. baumannii and K. pneumoniae, compared with cefiderocol. Potent in vivo efficacy was observed in a murine systemic infection model. Furthermore, the physicochemical stability of 86b in fluid medium at pH 6-8 was enhanced. 86b also reduced potential the risk of allergy owing to the quaternary ammonium linker. The improved properties of 86b supported its further research and development.

Dynamic Effect of ß-Lactam Antibiotic Inactivation Due to the Inter- and Intraspecies Interaction of Drug-Resistant Microbes.

The presence of ß-lactamase positive microorganisms imparts a pharmacological effect on a variety of organisms that can impact drug efficacy by influencing the function or composition of bacteria. Although studies to assess dynamic intra- and interspecies communication with bacterial communities exist, the efficacy of drug treatment and quantitative assessment of multiorganism response is not well understood due to the lack of technological advances that can be used to study coculture interactions in a dynamic format. In this study, we investigate how ß-lactamase positive microorganisms can neutralize the effect of ß-lactam antibiotics in a dynamic format at the inter- and intraspecies level using microbial bead technology. Three interactive models for the biological compartmentalization of organisms were demonstrated to evaluate the effect of ß-lactam antibiotics on coculture systems. Our model at the intraspecies level attempts to mimic the biofilm matrix more closely as a community-level feature of microorganisms, which acknowledges the impact of nondrug-resistant species in shaping the dynamic response. In particular, the results of intraspecies studies are highly supportive of the biofilm mode of bacterial growth, which can provide structural support and protect the bacteria from an assault on host or environmental factors. Our findings also indicate that ß-lactamase positive bacteria can neutralize the cytotoxic effect of ß-lactam antibiotics at the interspecies level when cocultured with cancer cells. Results were validated using ß-lactamase positive bacteria isolated from environmental niches, which can trigger phenotypical alteration of ß-lactams when cocultured with other organisms. Our compartmentalization strategy acts as an independent ecosystem and provides a new avenue for multiscale studies to assess intra- and interspecies interactions.

Novel Benzotriazole-ß-lactam Derivatives as Antimalarial Agents: Design, Synthesis, Biological Evaluation and Molecular Docking Studies.

Many people around the world suffer from malaria, especially in tropical or subtropical regions. While malaria medications have shown success in treating malaria, there is still a problem with resistance to these drugs. Herein, we designed and synthesized some structurally novel benzotriazole-ß-lactams using 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid as a key intermediate. To synthesize the target molecules, the ketene-imine cycloaddition reaction was employed. First, The reaction of 1H-benzo[d][1,2,3]triazole with 2-bromoacetic acid in aqueous sodium hydroxide yielded 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid. Then, the treatment of 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid with tosyl chloride, triethyl amine, and Schiff base provided new ß-lactams in good to moderate yields.The formation of all cycloadducts was confirmed by elemental analysis, FT-IR, NMR and mass spectral data. Moreover, X-ray crystallography was used to determine the relative stereochemistry of 4a compound. The in vitro antimalarial activity test was conducted for each compound against P. falciparum K1. The IC50 values ranged from 5.56 to 25.65 µM. A cytotoxicity profile of the compounds at 200 µM final concentration revealed suitable selectivity of the compounds for malaria treatment. Furthermore, the docking study was carried out for each compound into the P. falciparum dihydrofolate reductase enzyme (PfDHFR) binding site to analyze their possible binding orientation in the active site.

Cheminformatics identification of phenolics as modulators of penicillin-binding protein-3 of Pseudomonas aeruginosa towards interventive antibacterial therapy.

Antibacterial resistance to ß-lactams in microorganisms has been attributed majorly to alterations in penicillin-binding proteins (PBPs) coupled with ß-lactams' inactivation by ß-lactamase. Consequently, the identification of a novel class of therapeutics with improved modulatory action on the PBPs is imperative and plant secondary metabolites, including phenolics, have found relevance in this regard. For the first time in this study, the over 10,000 phenolics currently known were computationally evaluated against PBP3 of Pseudomonas aeruginosa, a superbug implicated in several nosocomial infections. In doing this, a library of phenolics with an affinity for PBP3 of P. aeruginosa was screened using structure-activity relationship-based pharmacophore and molecular docking approaches. Subsequent thermodynamic screening of the top five phenolics with higher docking scores, more drug-likeness attributes, and feasible synthetic accessibility was achieved through a 120 ns molecular dynamic (MD) simulation. Four of the top five hits had higher binding free energy than cefotaxime (-18.72 kcal/mol), with catechin-3-rhamside having the highest affinity (-28.99 kcal/mol). All the hits were stable at the active site of the PBP3, with catechin-3-rhamside being the most stable (2.14 Å), and established important interactions with Ser294, implicated in the catalytic activity of PBP3. Also, PBP3 became more compact with less fluctuation of the active site amino acid residues following the binding of the hits. These observations are indicative of the potential of the test compounds as PBP3 inhibitors, with catechin-3-rhamside being the most prominent of the compounds that could be further improved for enhanced druggability against PBP3 in vitro and in vivo.Communicated by Ramaswamy H. Sarma.

Chemical stability and physical compatibility of meropenem in admixtures for continuous and extended intravenous infusions / Estabilidad química y compatibilidad física de meropenem en mezclas para infusión intravenosa continua y extendida

Introduction: Prolonged intravenous infusion of beta-lactams increase the clinical cure rate compared to conventional administration in critical or septic patients. This study aimed to determine chemical stability and physical compatibility of meropenem at conditions used in clinical practice to evaluate the stability of the preparation during its administration and the possibility of anticipated preparation. Methods: Admixtures in study were: (i) meropenem 6g in 0.9% sodium chloride (NS) in infusor of 2mL/h 50mL or 10mL/h 240mL; (ii) meropenem 1 or 2g in NS in infusion bag of 250mL. Temperatures of study were: (i) infusor: 4.5°C, 32°C or 12h at 4.5°C followed by 32°C; (ii) Infusion bag: 4.5°C, 24.5°C or 6h at 4.5°C followed by 24.5°C. Time of study was 5–6 days in infusor and 1 day in infusion bag. Chemical stability was evaluated by high performance liquid chromatography and physical compatibility by measuring pH and visual inspection. Results: Chemical stability and physical compatibility of meropenem in admixtures in infusors were reduced at high meropenem concentration and high temperature. Admixtures in infusion bag show chemical stability and physical compatibility for at least 1 day. Conclusion: Administration of meropenem 6g in infusion of 24h in 240mL of 0.9% NaCl in infusor of 10mL/h could be possible if the admixture is infused at 4.5°C. Extended infusion of meropenem 1 or 2g in 0.9% NaCl in infusion bag (250mL) in 3–4h is also feasible. Anticipated preparation of the admixtures in infusion bag is possible with a stability of 24h.(AU)

Introducción: La infusión intravenosa prolongada de beta-lactámicos aumenta la velocidad de curación clínica comparada con la administración convencional en pacientes críticos o sépticos. Este estudio tiene como objetivo determinar la estabilidad química y la compatibilidad física de meropenem en condiciones utilizadas en la práctica clínica para evaluar la estabilidad de la preparación durante su administración y la posibilidad de la preparación anticipada. Métodos: Las mezclas en estudio fueron: (I) meropenem 6g en cloruro sódico 0,9% (SN) en infusor de 2mL/h 50 mL o 10mL/h 240mL; (iii) meropenem 1 o 2g en SN en bolsa de infusión de 250mL. Las temperaturas de estudio fueron: (i) infusor: 4,5°C, 32°C o 12h a 4,5°c seguido de 32°C; (ii) bolsa de infusión: 4,5°C, 24,5°C o 6h a 4,5°c seguido de 24,5°C. El tiempo de estudio fue de 5-6 días en infusor y 1 día en bolsa de infusión. Se evaluó la estabilidad química mediante cromatografía líquida de alta resolución y la compatibilidad física por medida de pH e inspección visual. Resultados: La estabilidad química y la compatibilidad física de meropenem en las mezclas en infusores disminuyeron al aumentar la concentración de meropenem y la temperatura. Las mezclas en bolsas de infusión mostraron estabilidad química y compatibilidad física durante al menos 1 día. Conclusión: La administración de meropenem 6g en infusión de 24h en 240 mL de cloruro sódico 0,9% en infusor de 10ml/h podría ser posible si la mezcla es administrada a 4,5°C. La infusión extendida de 1 o 2g en cloruro sódico 0,9% en bolsa de infusión (250 mL) en 3-4h es también viable. Puede realizarse la preparación anticipada de mezclas de meropenem en bolsas de infusión con una estabilidad de 1 día.(AU)

Penicillin-binding protein (PBP) inhibitor development: A 10-year chemical perspective.

Bacterial cell wall formation is essential for cellular survival and morphogenesis. The peptidoglycan (PG), a heteropolymer that surrounds the bacterial membrane, is a key component of the cell wall, and its multistep biosynthetic process is an attractive antibacterial development target. Penicillin-binding proteins (PBPs) are responsible for cross-linking PG stem peptides, and their central role in bacterial cell wall synthesis has made them the target of successful antibiotics, including ß-lactams, that have been used worldwide for decades. Following the discovery of penicillin, several other compounds with antibiotic activity have been discovered and, since then, have saved millions of lives. However, since pathogens inevitably become resistant to antibiotics, the search for new active compounds is continuous. The present review highlights the ongoing development of inhibitors acting mainly in the transpeptidase domain of PBPs with potential therapeutic applications for the development of new antibiotic agents. Both the critical aspects of the strategy, design, and structure-activity relationships (SAR) are discussed, covering the main published articles over the last 10 years. Some of the molecules described display activities against main bacterial pathogens and could open avenues toward the development of new, efficient antibacterial drugs.

The directed evolution of NDM-1.

ß-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward ß-lactam antibiotics is the production of ß-lactamases. These enzymes are capable of hydrolyzing the ß-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- ß-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- ß-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 ß-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

Synthesis by diastereomeric resolution, biochemical evaluation and molecular modelling of chiral 3-hydroxyl b-lactam microtubule-targeting agents for the treatment of triple negative breast and chemoresistant colorectal cancers.

The synthesis and biochemical activity of a series of chiral trans 3-hydroxyl ß-lactams targeting tubulin is described. Synthesis of the series of enantiopure ß-lactams was achieved using chiral derivatising reagent N-Boc-l-proline. The absolute configuration was determined as 3S,4S for (+) enantiomer 4EN1 and 3R,4R for (-) enantiomer 4EN2. Antiproliferative studies identified chiral 3S,4S b-lactams with subnanomolar IC50 values across a range of cancer cell lines, improving potency with respect to the corresponding racemates. Fluoro-substituted (+)-(3S,4S)-4-(3-fluoro-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27EN1) was determined as the lead eutomer with dual antiproliferative activity in triple negative breast cancer cells (TNBC), and combretastatin A-4 resistant HT-29 colorectal cancer cells. IC50 values were in the range of 0.26-0.7 nM across four cell lines. Tubulin polymerisation assays, confocal microscopy and molecular modelling studies indicated that 3S,4S eutomers are microtubule destabilisers, while 3R,4R distomers have lower potency as microtubule destabilisers. 27EN1 demonstrated anti-mitotic and pro-apoptotic activity in MDA-MB-231 and HT-29 cells in addition to selective toxicity toward MCF-7 breast cancer versus non-tumorigenic MCF-10-2A cells. The related 3S,4S ß-lactam eutomer 4EN1 downregulated expression of key cell survival anti-apoptotic proteins Bcl-2 and Mcl-1 in MDA-MB-231 cells while 27EN1 downregulated Mcl-1 in HT-29 cells. Chiral ß-lactam 27EN1 will be further developed for treatment of TNBC and CA-4 resistant colorectal cancers.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-ß-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution.

Gram-negative bacteria producing metallo-ß-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the ß-lactam ring present in a broad range of antibiotics, such as N-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (kfast = 1.76 h-1, kslow = 0.108 h-1 for IMP-1, and kfast = 14.0 h-1 and kslow = 1.66 h-1 for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against ß-lactam antibiotics, and the inhibitory effect of thiol compounds.

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.

Substituted phenethylamine-based ß-lactam derivatives: Antimicrobial, anticancer, and ß-lactamase inhibitory properties.

In this study, a series of novel ß-lactam derivatives were synthesized with yields ranging from 41 % to 91 %, and their antimicrobial activities were investigated against bacterial and fungal isolates that cause nosocomial infections. The results revealed that the novel ß-lactam derivatives, especially compound 19, showed antibacterial activities ranging from 0.98 to 250 µg/mL. In contrast, the compounds showed no antifungal activity against fungal isolates. Following that, biochemical (Nitrocefin) investigation of compounds with antibacterial activity was carried out, as well as their effects on the ß-lactamase enzyme. According to the results, the compounds inhibited the ß-lactamase enzyme against bacterial isolates. Furthermore, the anticancer activity and toxicity profiles of ß-lactam derivatives were also studied against colorectal cancer (Caco-2), the most common type of cancer, and non-human dermal fibroblast cell line. The results revealed that compound 19 was shown the best anticancer activity and lowest toxicity profile among other compounds. Therefore, these compounds, especially compound 19, could be useful in the treatment of colorectal cancer and related nosocomial infections. Furthermore, the potential of being an antibiotic can be put forward by investigating the resistant mechanism and further pharmacological studies.

Cytotoxic Assessment of 3,3-Dichloro-ß-Lactams Prepared through Microwave-Assisted Benzylic C-H Activation from Benzyl-Tethered Trichloroacetamides Catalyzed by RuCl2(PPh3)3.

Natural and synthetic ß-lactam derivatives constitute an interesting class of compounds due to their diverse biological activity. Mostly used as antibiotics, they were also found to have antitubercular, anticancer and antidiabetic activities, among others. In this investigation, six new 3,3-dichloro-ß-lactams prepared in a previous work were evaluated for their hemolytic and cytotoxic properties. The results showed that the proposed compounds have non-hemolytic properties and exhibited an interesting cytotoxic activity toward squamous cell carcinoma (A431 cell line), which was highly dependent on the structure and concentration of these ß-lactams. Among the molecules tested, 2b was the most cytotoxic, with the lowest IC50 values (30-47 µg/mL) and a promising selectivity against the tumor cells compared with non-tumoral cells.

Exploring the Synthetic Potential of γ-Lactam Derivatives Obtained from a Multicomponent Reaction-Applications as Antiproliferative Agents.

A study on the reactivity of 3-amino α,ß-unsaturated γ-lactam derivatives obtained from a multicomponent reaction is presented. Key features of the substrates are the presence of an endocyclic α,ß-unsaturated amide moiety and an enamine functionality. Following different synthetic protocols, the functionalization at three different positions of the lactam core is achieved. In the presence of a soft base, under thermodynamic conditions, the functionalization at C-4 takes place where the substrates behave as enamines, while the use of a strong base, under kinetic conditions, leads to the formation of C-5-functionalized γ-lactams, in the presence of ethyl glyoxalate, through a highly diastereoselective vinylogous aldol reaction. Moreover, the nucleophilic addition of organometallic species allows the functionalization at C-3, through the imine tautomer, affording γ-lactams bearing tetrasubstituted stereocenters, where the substrates act as imine electrophiles. Taking into account the advantage of the presence of a chiral stereocenter in C-5 substituted γ-lactams, further diastereoselective transformations are also explored, leading to novel bicyclic substrates holding a fused γ and δ-lactam skeleton. Remarkably, an example of a highly stereoselective formal [3+3] cycloaddition reaction of chiral γ-lactam substrates is reported for the synthesis of 1,4-dihidropyridines, where a non-covalent attractive interaction of a carbonyl group with an electron-deficient arene seems to drive the stereoselectivity of the reaction to the exclusive formation of the cis isomer. In order to unambiguously determine the substitution pattern resulting from the diverse reactions, an extensive characterization of the substrates is detailed through 2D NMR and/or X-ray experiments. Likewise, applications of the substrates as antiproliferative agents against lung and ovarian cancer cells are also described.

LAM-1 from Lysobacter antibioticus: A potent zinc-dependent activity that inactivates ß-lactam antibiotics.

Resistance to ß-lactam antibiotics, including the "last-resort" carbapenems, has emerged as a major threat to global health. A major resistance mechanism employed by pathogens involves the use of metallo-ß-lactamases (MBLs), zinc-dependent enzymes that inactivate most of the ß-lactam antibiotics used to treat infections. Variants of MBLs are frequently discovered in clinical environments. However, an increasing number of such enzymes have been identified in microorganisms that are less impacted by human activities. Here, an MBL from Lysobacter antibioticus, isolated from the rhizosphere, has been shown to be highly active toward numerous ß-lactam antibiotics. Its activity is higher than that of some of the most effective MBLs linked to hospital-acquired antibiotic resistance and thus poses an interesting system to investigate evolutionary pressures that drive the emergence of such biocatalysts.

Inhibition of ALK2 with bicyclic pyridyllactams.

Activin receptor-like kinase 2 (ALK2) has been implicated as a key target in multiple rare diseases. Herein, we describe the design of a novel bicyclic lactam series of potent and selective ALK2 inhibitors. This manuscript details an improvement in potency of two orders of magnitude from the initial bicyclic structure as well as a two-fold improvement in cellular potency from the original monocyclic inhibitor. Furthermore, we provide a detailed strategy for progressing this project in the future.

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.

Mechanistic insight on the inhibition of D, D-carboxypeptidase from Mycobacterium tuberculosis by ß-lactam antibiotics: an ONIOM acylation study.

Mycobacterium tuberculosis cell wall is intricate and impermeable to many agents. A D, D-carboxypeptidase (DacB1) is one of the enzymes involved in the biosynthesis of cell wall peptidoglycan and catalyzes the terminal D-alanine cleavage from pentapeptide precursors. Catalytic activity and mechanism by which DacB1 functions is poorly understood. Herein, we investigated the acylation mechanism of DacB1 by ß-lactams using a 6-membered ring transition state model that involves a catalytic water molecule in the reaction pathway. The full transition states (TS) optimization plus frequency were achieved using the ONIOM (B3LYP/6-31 + G(d): AMBER) method. Subsequently, the activation free energies were computed via single-point calculations on fully optimized structures using B3LYP/6-311++(d,p): AMBER and M06-2X/6-311++(d,p): AMBER with an electronic embedding scheme. The 6-membered ring transition state is an effective model to examine the inactivation of DacB1 via acylation by ß-lactams antibiotics (imipenem, meropenem, and faropenem) in the presence of the catalytic water. The ΔG# values obtained suggest that the nucleophilic attack on the carbonyl carbon is the rate-limiting step with 13.62, 19.60 and 30.29 kcal mol-1 for Imi-DacB1, Mero-DacB1 and Faro-DacB1, respectively. The electrostatic potential (ESP) and natural bond orbital (NBO) analysis provided significant electronic details of the electron-rich region and charge delocalization, respectively, based on the concerted 6-membered ring transition state. The stabilization energies of charge transfer within the catalytic reaction pathway concurred with the obtained activation free energies. The outcomes of this study provide important molecular insight into the inactivation of D, D-carboxypeptidase by ß-lactams.Communicated by Ramaswamy H. Sarma.

A multiscale approach to predict the binding mode of metallo beta-lactamase inhibitors.

Antibiotic resistance is a major threat to global public health. ß-lactamases, which catalyze breakdown of ß-lactam antibiotics, are a principal cause. Metallo ß-lactamases (MBLs) represent a particular challenge because they hydrolyze almost all ß-lactams and to date no MBL inhibitor has been approved for clinical use. Molecular simulations can aid drug discovery, for example, predicting inhibitor complexes, but empirical molecular mechanics (MM) methods often perform poorly for metalloproteins. Here we present a multiscale approach to model thiol inhibitor binding to IMP-1, a clinically important MBL containing two catalytic zinc ions, and predict the binding mode of a 2-mercaptomethyl thiazolidine (MMTZ) inhibitor. Inhibitors were first docked into the IMP-1 active site, testing different docking programs and scoring functions on multiple crystal structures. Complexes were then subjected to molecular dynamics (MD) simulations and subsequently refined through QM/MM optimization with a density functional theory (DFT) method, B3LYP/6-31G(d), increasing the accuracy of the method with successive steps. This workflow was tested on two IMP-1:MMTZ complexes, for which it reproduced crystallographically observed binding, and applied to predict the binding mode of a third MMTZ inhibitor for which a complex structure was crystallographically intractable. We also tested a 12-6-4 nonbonded interaction model in MD simulations and optimization with a SCC-DFTB QM/MM approach. The results show the limitations of empirical models for treating these systems and indicate the need for higher level calculations, for example, DFT/MM, for reliable structural predictions. This study demonstrates a reliable computational pipeline that can be applied to inhibitor design for MBLs and other zinc-metalloenzyme systems.