Structure-guided optimization of D-captopril for discovery of potent NDM-1 inhibitors.

ß-lactam antibiotics have long been the mainstay for the treatment of bacterial infections. New Delhi metallo-ß-lactamase 1 (NDM-1) is able to hydrolyze nearly all ß-lactam antibiotics and even clinically used serine-ß-lactamase inhibitors. The wide and rapid spreading of NDM-1 gene among pathogenic bacteria has attracted extensive attention, therefore high potency NDM-1 inhibitors are urgently needed. Here we report a series of structure-guided design of D-captopril derivatives that can inhibit the activity of NDM-1 in vitro and at cellular levels. Structural comparison indicates the mechanisms of inhibition enhancement and provides insights for further inhibitor optimization.

Bioisosteric investigation of ebselen: Synthesis and in vitro characterization of 1,2-benzisothiazol-3(2H)-one derivatives as potent New Delhi metallo-ß-lactamase inhibitors.

Carbapenem-resistant Enterobacteriaceae (CRE) producing New Delhi metallo-ß-lactamase (NDM-1) cause untreatable bacterial infections, posing a significant threat to human health. In the present study, by employing the concept of bioisosteric replacement of the selenium moiety of ebselen, we have designed, synthesized and characterized a small compound library of 2-substituted 1,2-benzisothiazol-3(2H)-one derivatives and related compounds for evaluating their cytotoxicity and synergistic activity in combination with meropenem against the E. coli Tg1 (NDM-1) strain. The most promising compound 3a demonstrated potent synergistic activity against a panel of clinically isolated NDM-1 positive CRE strains with FICI as low as 0.09. Moreover, its IC50 value and inhibition mechanism were also confirmed by using the enzyme inhibition assay and the ESI-MS analysis respectively. Importantly, compound 3a has acceptable toxicity and is not a PAINS. Because of its structural simplicity and potent synergistic activity in combination with meropenem, we propose that compound 3a may be a promising meropenem adjuvant and a new series of such compounds may worth further investigations.

Diversity and Proliferation of Metallo-ß-Lactamases: a Clarion Call for Clinically Effective Metallo-ß-Lactamase Inhibitors.

The worldwide proliferation of life-threatening metallo-ß-lactamase (MBL)-producing Gram-negative bacteria is a serious concern to public health. MBLs are compromising the therapeutic efficacies of ß-lactams, particularly carbapenems, which are last-resort antibiotics indicated for various multidrug-resistant bacterial infections. Inhibition of enzymes mediating antibiotic resistance in bacteria is one of the major promising means for overcoming bacterial resistance. Compounds having potential MBL-inhibitory activity have been reported, but none are currently under clinical trials. The need for developing safe and efficient MBL inhibitors (MBLIs) is obvious, particularly with the continuous spread of MBLs worldwide. In this review, the emergence and escalation of MBLs in Gram-negative bacteria are discussed. The relationships between different class B ß-lactamases identified up to 2017 are represented by a phylogenetic tree and summarized. In addition, approved and/or clinical-phase serine ß-lactamase inhibitors are recapitulated to reflect the successful advances made in developing class A ß-lactamase inhibitors. Reported MBLIs, their inhibitory properties, and their purported modes of inhibition are delineated. Insights into structural variations of MBLs and the challenges involved in developing potent MBLIs are also elucidated and discussed. Currently, natural products and MBL-resistant ß-lactam analogues are the most promising agents that can become clinically efficient MBLIs. A deeper comprehension of the mechanisms of action and activity spectra of the various MBLs and their inhibitors will serve as a bedrock for further investigations that can result in clinically useful MBLIs to curb this global menace.

Dithiocarbamates: Efficient metallo-ß-lactamase inhibitors with good antibacterial activity when combined with meropenem.

The activity of ß-lactam antibiotics is compromised by metallo-ß-lactamases (MBLs). Herein, a series of dithiocarbamate derivatives were designed and synthesized. Their antibacterial activities were tested in combination with meropenem (MEM) against several MBL (NDM and IMP type)-producing clinical isolates. Clinical isolates harboring NDM-1 and IMP-4 became susceptible to MEM when it was combined with dithiocarbamate compounds 4a, 4b or 4f synthesized in this work. Compounds 4a and 4b increased the effectiveness of MEM by up to 2560 times against strains. In vitro bactericidal dynamics tests showed that bacteria died within 24 h when they were treated with compound 4f + MEM. Compounds 4a, 4b and 4f were non-hemolytic and exhibited low toxicity toward HeLa cells in vitro. These data show that compounds containing dithiocarbamate functional group may be helpful in the development of MBL inhibitors.

Discovery of isatin-ß-methyldithiocarbazate derivatives as New Delhi metallo- ß-lactamase-1 (NDM-1) inhibitors against NDM-1 producing clinical isolates.

New Delhi metallo-ß-lactamase-1 (NDM-1) poses a threat to public health due to its capability to hydrolyze nearly all ß-lactam antibiotics, leaving limited treatment options for NDM-1 positive pathogens. Regrettably, there are presently no effective NDM-1 inhibitors in clinical use. This compels us to seek new compounds to combat multi-drug resistant bacterial infections (MDR). In our study, Zndm19 was identified as a new NDM-1 inhibitor through virtual screening and an NDM-1 enzyme activity inhibition assay. Subsequently, we employed the checkerboard method, time-killing assay, and combined disk test to investigate the synergistic bactericidal efficacy of Zndm19 in combination with meropenem (MEM). Meanwhile, molecular docking and site-directed mutagenesis were conducted to uncover the crucial amino acid residues engaged in Zndm19 binding. Finally, we established a mice peritonitis infection model to assess the synergistic effect of Zndm19 and MEM in vivo. Our findings demonstrated that 16 µg/mL of Zndm19 inhibited NDM-1 activity without affecting NDM-1 expression, restoring the bactericidal activity of MEM against NDM-1-positive Escherichia coli in vitro. Furthermore, MET-67, ASP-124, HIS-189, and HIS-250 amino acid residues constituted the active site of Zndm19 in NDM-1. Importantly, this combination therapy exhibited synergistic anti-infection activity in the mice peritonitis infection model, leading to an approximate 60% increase in survival rates and reduction of tissue bacterial load, effectively combating bacterial infection in vivo. In summary, our research validates that the synthetic novel NDM-1 inhibitor Zndm19 holds promise as a drug to treat drug-resistant bacterial infections, especially those harboring NDM-1.

1,2,4-Triazole-3-Thione Analogues with a 2-Ethylbenzoic Acid at Position 4 as VIM-type Metallo-ß-Lactamase Inhibitors.

Metallo-ß-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need. We previously reported several series of compounds based on the 1,2,4-triazole-3-thione scaffold. In particular, Schiff bases formed between diversely 5-substituted-4-amino compounds and 2-carboxybenzaldehyde were broad-spectrum inhibitors of VIM-type, NDM-1 and IMP-1 MBLs. Unfortunately, these compounds were unable to restore antibiotic susceptibility of MBL-producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we synthesized and characterized compounds where the hydrazone-like bond of the Schiff base analogues was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM-1 and IMP-1, but showed a significantly better activity on VIM-type enzymes, with Ki values in the µM to sub-µM range. The resolution of the crystallographic structure of VIM-2 in complex with one of the best inhibitors yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the ß-lactam susceptibility of VIM-type-producing E. coli laboratory strains and also of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cancer cells at high concentration, thus showing promising safety.

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.

Molecular docking studies, in-silico ADMET predictions and synthesis of novel PEGA-nucleosides as antimicrobial agents targeting class B1 metallo-ß-lactamases.

Class B1 metallo-ß-lactamases (MBLs) are metalloenzymes found in drug resistant bacteria. The enzyme requires zinc ions, along with conserved amino acid coordination for nucleophilic attack of the lactam ring to induce hydrolysis and inactivation of ß-lactam and some carbapenem antibiotics. To this date there are no clinically relevant class B1 MBL inhibitors, however L-captopril has shown significant results against NDM-1, the most difficult MBL to inhibit. Herein, we report the synthesis and evaluation of novel nucleoside analogues modified with polyethylene glycolamino (PEGA) as potential inhibitors for class B1 MBLs. Molecular dynamics simulations, using internal coordinate mechanics (ICM) algorithm, were performed on subclass B1 enzyme complex models screened with twenty-one possible PEGA-nucleosides. Analogue A, 3'-deoxy-3'-(2-(2-hydroxyethoxy)ethanamino)-ß-D-xylofuranosyluracil showed superior binding, with high specificity to the conserved zinc ions in the class B1 MBL active site by utilizing key ß-lactam mimic points in the uridine nucleobase. The PEGA moiety showed chelating activity with zinc and disrupted the metal-binding amino acid geometry. In all subclass B1 proteins tested, analogue A had the most effective inhibition when compared to penicillin or L-captopril. Chemical synthesis was performed by condensation of the corresponding keto ribonucleoside with PEGA, followed by enantioselective reduction of the formed imine to produce the amino derivative with desired configuration. Pharmacokinetic and pharmacodynamic screenings revealed that PEGA-pyrimidine nucleosides are not toxic, nor violate Lipinski's rules. These results suggested that analogue A can be proposed as a potential metalloenzyme inhibitor against the widespread antibiotic resistant bacteria and is worth further in vitro and in vivo investigations.

H2depda: An acyclic adjuvant potentiates meropenem activity in vitro against metallo-ß-lactamase-producing enterobacterales.

Metallo-ß-lactamase (MBL)-producing carbapenem-resistant Enterobacterales (CRE) pose an emerging threat to public health worldwide. An effective inhibitor of MBLs is therefore urgently needed for clinical use. In this study, two acyclic pyridine-containing ligands, H2dedpa and compound 8, were discovered with excellent activities when combined with meropenem (MEM) against MBL (blaNDM and blaIMP)-producing clinical isolates, including Escherichia coli, Citrobacter freundii, Proteus mirabilis, Enterobacter cloacae and Klebsiella pneumoniae. In particular, these two compounds improved the activity of MEM against E. coli harboring the blaNDM-4 gene by nearly 40,960 times. H2dedpa (IC50 = 0.17 ±â€¯0.04 µM) and compound 8 (IC50 = 0.04 ±â€¯0.02 µM) showed higher inhibitory activity against blaNDM-1 enzyme than the positive control ethylenediaminetetraacetic acid (EDTA, IC50 = 28.84 ±â€¯0.70 µM). A sterilization kinetics experiment showed that H2dedpain combined with MEM could kill 99.9% of bacteria within 24 h H2dedpa and compound 8 are therefore promising MBL inhibitors.

Impact of Pyridyl Moieties on the Inhibitory Properties of Prominent Acyclic Metal Chelators Against Metallo-ß-Lactamase-Producing Enterobacteriaceae: Investigating the Molecular Basis of Acyclic Metal Chelators' Activity.

Carbapenem-resistant Enterobacteriaceae (CREs)-mediated infections remain a huge public health concern. CREs produce enzymes such as metallo-ß-lactamases (MBLs), which inactivate ß-lactam antibiotics. Hence, developing efficient molecules capable of inhibiting these enzymes remains a way forward to overcoming this phenomenon. In this study, we demonstrate that pyridyl moieties favor the inhibitory activity of cyclic metal-chelating agents through in vitro screening, molecular modeling, and docking assays. Di-(2-picolyl) amine and tris-(2-picolyl) amine exhibited great efficacy against different types of MBLs and strong binding affinity for NDM-1, whereas 2-picolyl amine did not show activity at a concentration of 64 mg/L in combination with meropenem; it further showed the lowest binding affinity from computational molecular analysis, commensurating with the in vitro screening assays. The findings revealed that the pyridyl group plays a vital role in the inhibitory activity of the tested molecules against CREs and should be exploited as potential MBL inhibitors.

Approaches for the discovery of metallo-ß-lactamase inhibitors: A review.

After more than 80 years of development, ß-lactam drugs have become the most widely used high-efficiency, low-toxic broad-spectrum antibacterial drugs. However, with the widespread use and even abuse of those drugs, the resistance of major pathogens to ß-lactam drugs has increased over years, which has become a thorny problem to the public health. A common mechanism of the resistance to ß-lactams is the producing of ß-lactamases, which can hydrolyze the ß-lactam ring and inactivate these drugs. Metallo-ß-lactamases (MBLs) are one kind of ß-lactamases that require metal ions for their catalytic activities. Although it is a well-known strategy to recover the efficacy of ß-lactams by the combination of ß-lactamase inhibitors, there are still no MBL inhibitors that can be used in clinical practice. Therefore, it is urgent to develop MBL inhibitors. This review outlines the currently discovered MBL inhibitors with an emphasis on various strategies and approaches taken to discover MBL inhibitors, which may lead to diverse classes of inhibitors. Recent progress, particularly new screening methods, and the rational design of potent MBL inhibitors are discussed.

Metallo-ß-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding.

Bacterial resistance is compromising the use of ß-lactam antibiotics including carbapenems. The main resistance mechanism against ß-lactams is hydrolysis of the ß-lactam ring mediated by serine- or metallo-ß-lactamases (MBLs). Although several inhibitors of MBLs have been reported, none has been developed into a clinically useful inhibitor. Mercaptocarboxylic acids are among the most prominent scaffolds reported as MBL inhibitors. In this study, the carboxylate group of mercaptocarboxylic acids was replaced with bioisosteric groups like phosphonate esters, phosphonic acids and NH-tetrazoles. The influence of the replacement on the bioactivity and inhibitor binding was evaluated. A series of bioisosteres of previously reported inhibitors was synthesized and evaluated against the MBLs VIM-2, NDM-1 and GIM-1. The most active inhibitors combined a mercapto group and a phosphonate ester or acid, with two/three carbon chains connecting a phenyl group. Surprisingly, also compounds containing thioacetate groups instead of thiols showed low IC50 values. High-resolution crystal structures of three inhibitors in complex with VIM-2 revealed hydrophobic interactions for the diethyl groups in the phosphonate ester (inhibitor 2b), the mercapto bridging the two active site zinc ions, and tight stacking of the benzene ring to the inhibitor between Phe62, Tyr67, Arg228 and His263. The inhibitors show reduced enzyme activity in Escherichia coli cells harboring MBL. The obtained results will be useful for further structural guided design of MBL inhibitors.

Evaluation of the total MBL confirm kit (ROSCO) for detection of metallo-ß-lactamases in Pseudomonas aeruginosa and Acinetobacter baumannii.

Phenotypic tests for carbapenemase production in Pseudomonas aeruginosa and Acinetobacter baumannii have been associated with unspecific metallo-ß-lactamase (MBL) inhibitor activity in synergy tests and low positive predictive value. In this study, a collection of well-characterized P. aeruginosa and A. baumannii isolates was used to evaluate the inhibitor-based Total MBL Confirm Kit and the MBL Etest.