Novel partially reversible NDM-1 inhibitors based on the naturally occurring houttuynin.

Discovering novel NDM-1 inhibitors is an urgent task for treatment of 'superbug' infectious diseases. In this study, we found that naturally occurring houttuynin and its sulfonate derivatives might be effective NDM-1 inhibitors with novel mechanism, i.e. the attribute of partially covalent inhibition of sulfonate derivatives of houttuynin against NDM-1. Primary structure-activity relationship study showed that both the long aliphatic side chain and the warhead of aldehyde group are vital for the efficiency against NDM-1. The homologs with longer chains (SNH-2 to SNH-5) displayed stronger inhibitory activities with IC50 range of 1.1-1.5 µM, while the shorter chain the weaker inhibition. Further synergistic experiments in cell level confirmed that all these 4 compounds (at 32 µg/mL) recovered the antibacterial activity of meropenem (MER) against E. coli BL21/pET15b-blaNDM-1.

Structure-guided optimization of 1H-imidazole-2-carboxylic acid derivatives affording potent VIM-Type metallo-ß-lactamase inhibitors.

Production of metallo-ß-lactamases (MBLs) in bacterial pathogens is an important cause of resistance to the 'last-resort' carbapenem antibiotics. Development of effective MBL inhibitors to reverse carbapenem resistance in Gram-negative bacteria is still needed. We herein report X-ray structure-guided optimization of 1H-imidazole-2-carboxylic acid (ICA) derivatives by considering how to engage with the active-site flexible loops and improve penetration into Gram-negative bacteria. Structure-activity relationship studies revealed the importance of appropriate substituents at ICA 1-position to achieve potent inhibition to class B1 MBLs, particularly the Verona Integron-encoded MBLs (VIMs), mainly by involving ingenious interactions with the flexible active site loops as observed by crystallographic analyses. Of the tested ICA inhibitors, 55 displayed potent synergistic antibacterial activity with meropenem against engineered Escherichia coli strains and even intractable clinically isolated Pseudomonas aeruginosa producing VIM-2 MBL. The morphologic and internal structural changes of bacterial cells after treatment further demonstrated that 55 crossed the outer membrane and reversed the activity of meropenem. Moreover, 55 showed good pharmacokinetic and safety profile in vivo, which could be a potential candidate for combating VIM-mediated Gram-negative carbapenem resistance.

Nitroxoline and its derivatives are potent inhibitors of metallo-ß-lactamases.

Carbapenemases such as metallo-ß-lactamases (MBLs) are spreading among Gram-negative bacterial pathogens. Infections due to these multidrug-resistant bacteria constitute a major global health challenge. Therapeutic strategies against carbapenemase producing bacteria include ß-lactamase inhibitor combinations. Nitroxoline is a broad-spectrum antibiotic with restricted indication for urinary tract infections. In this study, we report on nitroxoline as an inhibitor of MBLs. We investigate the structure-activity relationships of nitroxoline derivatives considering in vitro MBL inhibitory potency in a fluorescence based assay using purified recombinant MBLs, NDM-1 and VIM-1. We investigated the most potent nitroxoline derivative in combination with imipenem against clinical isolates as well as transformants producing MBL by broth microdilution and time-kill kinetics. Our findings demonstrate that nitroxoline derivatives are potent MBL inhibitors and in combination with imipenem overcome MBL-mediated carbapenem resistance.

1,2,4-Triazole-3-thione compounds with a 4-ethyl alkyl/aryl sulfide substituent are broad-spectrum metallo-ß-lactamase inhibitors with re-sensitization activity.

Metallo-ß-lactamases (MBLs) are important contributors of Gram-negative bacteria resistance to ß-lactam antibiotics. MBLs are highly worrying because of their carbapenemase activity, their rapid spread in major human opportunistic pathogens while no clinically useful inhibitor is available yet. In this context, we are exploring the potential of compounds based on the 1,2,4-triazole-3-thione scaffold as an original ligand of the di-zinc active sites of MBLs, and diversely substituted at its positions 4 and 5. Here, we present a new series of compounds substituted at the 4-position by a thioether-containing alkyl chain with a carboxylic and/or an aryl group at its extremity. Several compounds showed broad-spectrum inhibition with Ki values in the µM to sub-µM range against VIM-type enzymes, NDM-1 and IMP-1. The presence of the sulfur and of the aryl group was important for the inhibitory activity and the binding mode of a few compounds in VIM-2 was revealed by X-ray crystallography. Importantly, in vitro antibacterial susceptibility assays showed that several inhibitors were able to potentiate the activity of meropenem on Klebsiella pneumoniae clinical isolates producing VIM-1 or VIM-4, with a potentiation effect of up to 16-fold. Finally, a selected compound was found to only moderately inhibit the di-zinc human glyoxalase II, and several showed no or only moderate toxicity toward several human cells, thus favourably completing a promising behaviour.

High-Throughput Crystallography Reveals Boron-Containing Inhibitors of a Penicillin-Binding Protein with Di- and Tricovalent Binding Modes.

The effectiveness of ß-lactam antibiotics is increasingly compromised by ß-lactamases. Boron-containing inhibitors are potent serine-ß-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) ß-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by ß-lactamase-driven resistance.

Efforts towards the inhibitor design for New Delhi metallo-beta-lactamase (NDM-1).

Antimicrobial stewardship is imperative when treating bacterial infections because the misuse and overuse of antibiotics have caused pathogens to develop life-threatening resistance mechanisms. The New Delhi metallo-beta-lactamase (NDM-1) is one of many enzymes that enable bacterial resistance. NDM-1 is a more recently discovered beta-lactamase with the ability to inactivate a wide range of beta-lactam antibiotics. Multiple NDM-1 inhibitors have been designed and tested; however, due to the complexity of the NDM-1 active site, there is currently no inhibitor on the market. Consequently, an infection caused by bacteria possessing the gene for the NDM-1 enzyme is a serious and potentially fatal complication. An abundance of research has been invested over the past decade in search of an NDM-1 inhibitor. This review aims to summarize various NDM-1 inhibitor designs that have been developed in recent years.

N-acylhydrazones confer inhibitory efficacy against New Delhi metallo-ß-lactamase-1.

The expression of ß-lactamases, especially metallo-ß-lactamases (MßLs) in bacteria is one of the main causes of drug resistance. In this work, an effective N-acylhydrazone scaffold as MßL inhibitor was constructed and characterized. The biological activity assays indicated that the synthesized N-acylhydrazones 1-11 preferentially inhibited MßL NDM-1, and 1 was found to be the most effective inhibitor with an IC50 of 1.2 µM. Analysis of IC50 data revealed a structure-activity relationship, which is that the pyridine and hydroxylbenzene substituents at 2-position improved inhibition of the compounds on NDM-1. ITC and enzyme kinetics assays suggested that it reversibly and competitively inhibited NDM-1 (Ki = 0.29 ± 0.05 µM). The synthesized N-acylhydrazones showed synergistic antibacterial activities with meropenem, reduced 4-16-fold MIC of meropenem on NDM-1- producing E. coli BL21 (DE3), while 1 restored 4-fold activity of meropenem on K. pneumonia expressing NDM-1 (NDM-K. pneumoniae). The mice experiments suggested that 1 combined meropenem to fight against NDM-K. pneumoniae infection in the spleen and liver. Cytotoxicity assays showed that 1 and 2 have low cytotoxicity. This study offered a new framework for the development of NDM-1 inhibitors.

Thiosemicarbazones exhibit inhibitory efficacy against New Delhi metallo-ß-lactamase-1 (NDM-1).

The superbug infection caused by metallo-ß-lactamases (MßLs) carrying drug-resistant bacteria, specifically, New Delhi metallo-ß-lactamase (NDM-1) has become an emerging threat. In an effort to develop novel inhibitors of NDM-1, thirteen thiosemicarbazones (1a-1m) were synthesized and assayed. The obtained molecules specifically inhibited NDM-1, with an IC50 in the range of 0.88-20.2 µM, and 1a and 1f were found to be the potent inhibitors (IC50 = 1.79 and 0.88 µM) using cefazolin as substrate. ITC and kinetic assays indicated that 1a irreversibly and non-competitively inhibited NDM-1 in vitro. Importantly, MIC assays revealed that these molecules by themselves can sterilize NDM-producing clinical isolates EC01 and EC08, exhibited 78-312-fold stronger activities than the cefazolin. MIC assays suggest that 1a (16 µg ml-1) has synergistic antimicrobial effect with ampicillin, cefazolin and meropenem on E. coli producing NDM-1, resulting in MICs of 4-32-, 4-32-, and 4-8-fold decrease, respectively. These studies indicate that the thiosemicarbazide is a valuable scaffold for the development of inhibitors of NDM-1 and NDM-1 carrying drug-resistant bacteria.

H2dpa derivatives containing pentadentate ligands: An acyclic adjuvant potentiates meropenem activity in vitro and in vivo against metallo-ß-lactamase-producing Enterobacterales.

The emergence and dissemination of metallo-ß-lactamases (MBLs) producing Enterobacterales is a great concern for public health due to the limited therapeutic options. No MBL inhibitors are currently available in clinical practice. Herein, we synthesized a series of H2dpa derivatives containing pentadentate-chelating ligands and evaluated their inhibitory activity against MBLs. Related compounds inhibited clinically relevant MBLs (Imipenemase, New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase) with IC50 values of 1-4.9 µM. In vitro, the most promising compounds, 5b and 5c, which had a chiral methyl at the acid adjacent to 5a, demonstrated potent synergistic activity against engineered strains, with fractional inhibitory concentration index values as low as 0.07-0.18. The addition of 5b and 5c restored meropenem efficacy against 42 MBL-producing Enterobacterales and Pseudomonas aeruginosa to satisfactory clinical levels. In addition, safety tests revealed that 5b/5c showed no toxicity in red blood cells, cell lines or mouse model. Further studies demonstrated that compounds 5b and 5c were non-competitive MBL inhibitors. In vivo compounds 5b and 5c potentiated meropenem efficacy and increased the survival rate from 0 to at least 83% in mice with sepsis caused by an NDM-1-positive clinical strain. The activity of the compounds exhibited consistency at the molecular, cellular, and in vivo levels. These data indicated that H2dpa derivatives 5b and 5c containing pentadentate-chelating ligands may be worthy of further study.

Discovery of 2-Sulfinyl-Diazabicyclooctane Derivatives, Potential Oral ß-Lactamase Inhibitors for Infections Caused by Serine ß-Lactamase-Producing Enterobacterales.

Coadministration of ß-lactam and ß-lactamase inhibitor (BLI) is one of the well-established therapeutic measures for bacterial infections caused by ß-lactam-resistant Gram-negative bacteria, whereas we have only two options for orally active BLI, clavulanic acid and sulbactam. Furthermore, these BLIs are losing their clinical usefulness because of the spread of new ß-lactamases, including extended-spectrum ß-lactamases (ESBLs) belonging to class A ß-lactamases, class C and D ß-lactamases, and carbapenemases, which are hardly or not inhibited by these classical BLIs. From the viewpoints of medical cost and burden of healthcare personnel, oral therapy offers many advantages. In our search for novel diazabicyclooctane (DBO) BLIs possessing a thio-functional group at the C2 position, we discovered a 2-sulfinyl-DBO derivative (2), which restores the antibacterial activities of an orally available third-generation cephalosporin, ceftibuten (CTB), against various serine ß-lactamase-producing strains including carbapenem-resistant Enterobacteriaceae (CRE). It can be orally absorbed via the ester prodrug modification and exhibits in vivo efficacy in a combination with CTB.

RhII -Catalyzed De-symmetrization of Ethane-1,2-dithiol and Propane-1,3-dithiol Yields Metallo-ß-lactamase Inhibitors.

Diversity-oriented synthesis (DOS) is a rich source for novel lead structures in Medicinal Chemistry. In this study, we present a DOS-compatible method for synthesis of compounds bearing a free thiol moiety. The procedure relies on Rh(II)-catalyzed coupling of dithiols to diazo building blocks. The synthetized library was probed against metallo-ß-lactamases (MBLs) NDM-1 and VIM-1. Biochemical and biological evaluation led to identification of novel potent MBL inhibitors with antibiotic adjuvant activity.

Discovery of VNRX-7145 (VNRX-5236 Etzadroxil): An Orally Bioavailable ß-Lactamase Inhibitor for Enterobacterales Expressing Ambler Class A, C, and D Enzymes.

A major antimicrobial resistance mechanism in Gram-negative bacteria is the production of ß-lactamase enzymes. The increasing emergence of ß-lactamase-producing multi-drug-resistant "superbugs" has resulted in increases in costly hospital Emergency Department (ED) visits and hospitalizations due to the requirement for parenteral antibiotic therapy for infections caused by these difficult-to-treat bacteria. To address the lack of outpatient treatment, we initiated an iterative program combining medicinal chemistry, biochemical testing, microbiological profiling, and evaluation of oral pharmacokinetics. Lead optimization focusing on multiple smaller, more lipophilic active compounds, followed by an exploration of oral bioavailability of a variety of their respective prodrugs, provided 36 (VNRX-7145/VNRX-5236 etzadroxil), the prodrug of the boronic acid-containing ß-lactamase inhibitor 5 (VNRX-5236). In vitro and in vivo studies demonstrated that 5 restored the activity of the oral cephalosporin antibiotic ceftibuten against Enterobacterales expressing Ambler class A extended-spectrum ß-lactamases, class A carbapenemases, class C cephalosporinases, and class D oxacillinases.

Metallo-ß-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design.

Antimicrobial resistance is one of the major problems in current practical medicine. The spread of genes coding for resistance determinants among bacteria challenges the use of approved antibiotics, narrowing the options for treatment. Resistance to carbapenems, last resort antibiotics, is a major concern. Metallo-ß-lactamases (MBLs) hydrolyze carbapenems, penicillins, and cephalosporins, becoming central to this problem. These enzymes diverge with respect to serine-ß-lactamases by exhibiting a different fold, active site, and catalytic features. Elucidating their catalytic mechanism has been a big challenge in the field that has limited the development of useful inhibitors. This review covers exhaustively the details of the active-site chemistries, the diversity of MBL alleles, the catalytic mechanism against different substrates, and how this information has helped developing inhibitors. We also discuss here different aspects critical to understand the success of MBLs in conferring resistance: the molecular determinants of their dissemination, their cell physiology, from the biogenesis to the processing involved in the transit to the periplasm, and the uptake of the Zn(II) ions upon metal starvation conditions, such as those encountered during an infection. In this regard, the chemical, biochemical and microbiological aspects provide an integrative view of the current knowledge of MBLs.

Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of Imipenemase-Type Metallo-ß-Lactamases.

In an attempt to exploit the hydrolytic mechanism by which ß-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-ß-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the ß-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity.

Dipyridyl-substituted thiosemicarbazone as a potent broad-spectrum inhibitor of metallo-ß-lactamases.

To combat the superbug infection caused by metallo-ß-lactamases (MßLs), a dipyridyl-substituted thiosemicarbazone (DpC), was identified to be the broad-spectrum inhibitor of MßLs (NDM-1, VIM-2, IMP-1, ImiS, L1), with an IC50 value in the range of 0.021-1.08 µM. It reversibly and competitively inhibited NDM-1 with a Ki value of 10.2 nM. DpC showed broad-spectrum antibacterial effect on clinical isolate K. pneumonia, CRE, VRE, CRPA and MRSA, with MIC value ranged from 16 to 32 µg/mL, and exhibited synergistic antibacterial effect with meropenem on MßLs-producing bacteria, resulting in a 2-16-, 2-8-, and 8-fold reduction in MIC of meropenem against EC-MßLs, EC01-EC24, K. pneumonia, respectively. Moreover, mice experiments showed that DpC also had synergistic antibacterial action with meropenem. In this work, DpC was identified to be a potent scaffold for the development of broad-spectrum inhibitors of MßLs.

Docking and antibacterial activity of novel nontoxic 5-arylidenepyrimidine-triones as inhibitors of NDM-1 and MetAP-1.

Background: Antibiotic resistance, which occurs through the action of metallo-ß-lactamases (NDM-1), is a serious problem in the treatment of infectious diseases. Therefore, the discovery of new NDM-1 inhibitors and promising antibacterial agents as inhibitors of alternative targets (MetAP-1) is important. Method & results: In this study, a virtual library of 5-arylidene barbituric acids was created and molecular docking was performed for identification of novel possible inhibitors of NDM-1 and MetAP-1. Antibacterial activity (agar well-diffusion assay) and cytotoxicity (alamarBlue assay) of perspective compounds were evaluated. Pharmacokinetic profiles and molecular properties were predicted. Conclusion: We have identified possible novel inhibitors of NDM-1 and MetAP-1 with bacteriostatic activity, most of which are not cytotoxic and have potential excellent drug-likeness properties.

Designing Inhibitors of ß-Lactamase Enzymes to Overcome Carbapenem Resistance in Gram-Negative Bacteria.

Ever since the first ß-lactam antibiotic, penicillin, was introduced into the clinic over 70 years ago, resistance has been observed because of the presence of ß-lactamase enzymes, which hydrolyze the ß-lactam ring of ß-lactam antibiotics. Early ß-lactamase enzymes were all of the serine ß-lactamase (SBL) type, but more recently, highly resistant Gram-negative strains have emerged in which metallo-ß-lactamase (MBL) enzymes are responsible for resistance. The two types of ß-lactamase enzymes are structurally and mechanistically different but serve the same purpose in bacteria. The SBLs use an active serine group as a nucleophile to attack the ß-lactamase ring, forming a covalent intermediate that is subsequently hydrolyzed. In contrast, the MBLs use a zinc ion to activate the ß-lactam toward nucleophilic attack by a hydroxide anion held between two zinc ions. In this Account, we review our recent contribution to the field of ß-lactamase inhibitor design in terms of both SBL and MBL inhibitors. We describe how we have approached these challenges from the particular perspective of a small biotechnology company, identifying new inhibitors when faced with either a paucity of starting points for medicinal chemistry (MBL inhibitors) or else an abundance of prior research necessitating a search for novelty, improvement, and differentiation (SBL inhibitors). During the journey from the beginning of lead optimization to successful identification of a preclinical candidate for development, we encountered and solved a range of issues. For example, in the MBL inhibitor series we were able to prevent metabolic cleavage of a glycinamide moiety by circulating amidases while still retaining the activity by converting the amino group into a guanidine. In the SBL inhibitor series, the structure-activity relationship led us to consider introducing a fluorine substituent adjacent to a urea functionality. At first sight this grouping would appear to be chemically unstable. However, deeper theoretical considerations suggested that this would not be the case, and in practice the compound is remarkably stable. Both examples serve to illustrate the importance of scientific insight and the necessity to explore speculative hypotheses as part of the creative medicinal chemistry process.

Discovery of 3-aryl substituted benzoxaboroles as broad-spectrum inhibitors of serine- and metallo-ß-lactamases.

The production of ß-lactamases represents the main cause of resistance to clinically important ß-lactam antibiotics. Boron containing compounds have been demonstrated as promising broad-spectrum ß-lactamase inhibitors to combat ß-lactam resistance. Here we report a series of 3-aryl substituted benzoxaborole derivatives, which manifested broad-spectrum inhibition to representative serine-ß-lactamases (SBLs) and metallo-ß-lactamases (MBLs). The most potent inhibitor 9f displayed an IC50 value of 86 nM to KPC-2 SBL and micromolar inhibitory activity towards other tested enzymes. Cell-based assays further revealed that 9f was able to significantly reduce the MICs of meropenem in clinically isolated KPC-2-producing bacterial strains and it showed no apparent toxicity in HEK293T cells.

Mechanistic Investigations of Metallo-ß-lactamase Inhibitors: Strong Zinc Binding Is Not Required for Potent Enzyme Inhibition*.

Metallo-ß-lactamases (MBLs) are zinc-dependent bacterial enzymes that inactivate essentially all classes of ß-lactam antibiotics including last-resort carbapenems. At present there are no clinically approved MBL inhibitors, and in order to develop such agents it is essential to understand their inhibitory mechanisms. Herein, we describe a comprehensive mechanistic study of a panel of structurally distinct MBL inhibitors reported in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration (IC50 ) for each inhibitor against MBLs belonging to the NDM and IMP families. In addition, the binding affinities of the inhibitors for Zn2+ , Ca2+  and Mg2+  were assessed by using isothermal titration calorimetry (ITC). We also compared the ability of the different inhibitors to resensitize a highly resistant MBL-expressing Escherichia coli strain to meropenem. These investigations reveal clear differences between the MBL inhibitors studied in terms of their IC50 value, metal binding ability, and capacity to synergize with meropenem. Notably, our studies demonstrate that potent MBL inhibition and synergy with meropenem are not explicitly dependent on the capacity of an inhibitor to strongly chelate zinc.

Development of sulfahydantoin derivatives as ß-lactamase inhibitors.

Sulfahydantoin-based molecules may provide a means to counteract antibiotic resistance, which is on the rise. These molecules may act as inhibitors of ß-lactamase enzymes, which are key in some resistance mechanisms. In this paper, we report on the synthesis of 6 novel sulfahydantoin derivatives by the key reaction of chlorosulfonyl isocyanate to form α-amino acid derived sulfamides, and their cyclization into sulfahydantoins. The synthesis is rapid and provides the target compounds in 8 steps. We investigated their potential as ß-lactamase inhibitors using two common Class A ß-lactamases, TEM-1 and the prevalent extended-spectrum TEM-15. Two compounds, 3 and 6, show substantial inhibition of the ß-lactamases with IC50 values between 130 and 510 µM and inferred Ki values between 32 and 55 µM.