Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1ß in Cellular and Animal Infection Models.

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1ß. Furthermore, inhibition of LasB-mediated IL-1ß activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.

Optimization of 1,2,4-Triazole-3-thiones toward Broad-Spectrum Metallo-ß-lactamase Inhibitors Showing Potent Synergistic Activity on VIM- and NDM-1-Producing Clinical Isolates.

Metallo-ß-lactamases (MBLs) contribute to the resistance of Gram-negative bacteria to carbapenems, last-resort antibiotics at hospital, and MBL inhibitors are urgently needed to preserve these important antibacterial drugs. Here, we describe a series of 1,2,4-triazole-3-thione-based inhibitors displaying an α-amino acid substituent, which amine was mono- or disubstituted by (hetero)aryl groups. Compounds disubstituted by certain nitrogen-containing heterocycles showed submicromolar activities against VIM-type enzymes and strong NDM-1 inhibition (Ki = 10-30 nM). Equilibrium dialysis, native mass spectrometry, isothermal calorimetry (ITC), and X-ray crystallography showed that the compounds inhibited both VIM-2 and NDM-1 at least partially by stripping the catalytic zinc ions. These inhibitors also displayed a very potent synergistic activity with meropenem (16- to 1000-fold minimum inhibitory concentration (MIC) reduction) against VIM-type- and NDM-1-producing ultraresistant clinical isolates, including Enterobacterales and Pseudomonas aeruginosa. Furthermore, selected compounds exhibited no or moderate toxicity toward HeLa cells, favorable absorption, distribution, metabolism, excretion (ADME) properties, and no or modest inhibition of several mammalian metalloenzymes.

1,2,4-Triazole-3-thione analogues with an arylakyl group at position 4 as metallo-ß-lactamase inhibitors.

Metallo-ß-lactamases (MBLs) represent an increasingly serious threat to public health because of their increased prevalence worldwide in relevant opportunistic Gram-negative pathogens. MBLs efficiently inactivate widely used and most valuable ß-lactam antibiotics, such as oxyiminocephalosporins (ceftriaxone, ceftazidime) and the last-resort carbapenems. To date, no MBL inhibitor has been approved for therapeutic applications. We are developing inhibitors characterized by a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported. Here, we present the synthesis and evaluation of a new series of compounds characterized by the presence of an arylalkyl substituent at position 4 of the triazole ring. The alkyl link was mainly an ethylene, but a few compounds without alkyl or with an alkyl group of various lengths up to a butyl chain were also synthesized. Some compounds in both sub-series were micromolar to submicromolar inhibitors of tested VIM-type MBLs. A few of them were broad-spectrum inhibitors, as they showed significant inhibitory activity on NDM-1 and, to a lesser extent, IMP-1. Among these, several inhibitors were able to significantly reduce the meropenem MIC on VIM-1- and VIM-4- producing clinical isolates by up to 16-fold. In addition, ACE inhibition was absent or moderate and one promising compound did not show toxicity toward HeLa cells at concentrations up to 250 µM. This series represents a promising basis for further exploration. Finally, molecular modelling of representative compounds in complex with VIM-2 was performed to study their binding mode.

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.

4-Alkyl-1,2,4-triazole-3-thione analogues as metallo-ß-lactamase inhibitors.

In Gram-negative bacteria, the major mechanism of resistance to ß-lactam antibiotics is the production of one or several ß-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-ß-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the µM to sub-µM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.