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Since the discovery of penicillin, the forerunner of the most widely used class of antibiotics ( i.e. β-lactams), natural compounds and their derivatives represented a major source of antibacterial therapeutic products whose availability enabled modern medical practices (invasive surgery, organ transplant, etc .). However, the relentless emergence of resistant bacteria is challenging the long-term efficacy of antibiotics, also decreasing their economic attractiveness for big pharma, leading to a significant decay in antibacterial development in the 21 st century and an increased use of last-resort drugs such as carbapenems or colistin. Indeed, bacteria evolved an arsenal of resistance mechanisms, leading to the emergence of totally-drug resistant isolates, already sporadically isolated among Gram-negative bacterial species. To face this deadly peril, it is fundamental to explore new ground-breaking approaches. In view of the significance of both β-lactam antibiotics and the production of one or more β-lactamases as the major resistance mechanism (especially in Gram-negative bacteria), we implemented an original approach to selectively deliver antibacterial zidovudine (AZT) exploiting the β-lactamase-mediated hydrolysis of a β-lactam-conjugate prodrug. The synthesis of the targeted pronucleosides was performed in 5-7 steps and based on an original Pd-catalyzed cross-coupling reaction. Enzymatic and microbiological evaluations were performed to evaluate the synthesized pronucleosides, yielding new insights into molecular recognition of β-lactamase enzymes. This approach would potentially allow a targeted and selective eradication of antibiotic-resistant β-lactamase-producing (opportunistic) pathogens, as the inactive prodrug is unable to harm the commensal microbial flora.

The circular bioeconomy is currently a promising model for repurposing natural sources; these sources include plants due to their abundance of bioactive compounds. This study evaluated the antimicrobial properties of a Chaetomorpha linum extract. Chaetomorpha linum is an invasive macroalga from the Orbetello Lagoon (Tuscany, Italy), which grows in nutrient-rich environments and has been forming extended mats since 2005. The biomass is mechanically harvested and treated as waste, consuming considerable manpower and financial resources. As a potential way to increase the value of such waste, this study found that C. linum extract (CLE) is a source of antimicrobial compounds. The phytochemical characterization of the extract revealed the predominant presence of palmitic acid, a fatty acid with known antimicrobial activity. Based on such findings, four bacterial species of high clinical relevance ( Enterococcus faecalis , Staphylococcus aureus , Pseudomonas aeruginosa , and Escherichia coli ) were tested, revealing a notable antibacterial activity of the extract on Enterococcus faecalis (MIC, 32 μg/mL). Computational analyses identified a potential Enterococcus faecalis molecular target for palmitic acid, offering molecular insights on the interaction. This study presents a comprehensive in vitro and in silico approach for drug and target discovery studies by repurposing C. linum as a source of antimicrobial bioactive compounds.

Antimicrobial resistance (AMR) represents one of the most challenging global Public Health issues, with an alarmingly increasing rate of attributable mortality. This scenario highlights the urgent need for innovative medicinal strategies showing activity on resistant isolates (especially, carbapenem-resistant Gram-negative bacteria, methicillin-resistant S. aureus, and vancomycin-resistant enterococci) yielding new approaches for the treatment of bacterial infections. We previously reported AlkylGuanidino Ureas (AGUs) with broad-spectrum antibacterial activity and a putative membrane-based mechanism of action. Herein, new tetra- and mono-guanidino derivatives were designed and synthesized to expand the structure-activity relationships (SARs) and, thereby, tested on the same panel of Gram-positive and Gram-negative bacteria. The membrane-active mechanism of selected compounds was then investigated through molecular dynamics (MD) on simulated bacterial membranes. In the end, the newly synthesized series, along with the whole library of compounds (more than 70) developed in the last decade, was tested in combination with subinhibitory concentrations of the last resort antibiotic colistin to assess putative synergistic or additive effects. Moreover, all the AGUs were subjected to cheminformatic and machine learning analyses to gain a deeper knowledge of the key features required for bioactivity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the work is partially supported by Lead Discovery Siena s.r.l. They declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Respiratory tract infections involving a variety of microorganisms such as viruses, bacteria, and fungi are a prominent cause of morbidity and mortality globally, exacerbating various pre-existing respiratory and non-respiratory conditions. Moreover, the ability of bacteria and viruses to coexist might impact the development and severity of lung infections, promoting bacterial colonization and subsequent disease exacerbation. Secondary bacterial infections following viral infections represent a complex challenge to be overcome from a therapeutic point of view. We report herein our efforts in the development of new bithiazole derivatives showing broad-spectrum antimicrobial activity against both viruses and bacteria. A series of 4-trifluoromethyl bithiazole analogues was synthesized and screened against selected viruses (hRVA16, EVD68, and ZIKV) and a panel of Gram-positive and Gram-negative bacteria. Among them, two promising broad-spectrum antimicrobial compounds (8a and 8j) have been identified: both compounds showed low micromolar activity against all tested viruses, 8a showed synergistic activity against E. coli and A. baumannii in the presence of a subinhibitory concentration of colistin, while 8j showed a broader spectrum of activity against Gram-positive and Gram-negative bacteria. Activity against antibiotic-resistant clinical isolates is also reported. Given the ever-increasing need to adequately address viral and bacterial infections or co-infections, this study paves the way for the development of new agents with broad antimicrobial properties and synergistic activity with common antivirals and antibacterials., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

The worldwide emergence and dissemination of Gram-negative bacteria expressing metallo-β-lactamases (MBLs) menace the efficacy of all β-lactam antibiotics, including carbapenems, a last-line treatment usually restricted to severe pneumonia and urinary tract infections. Nonetheless, no MBL inhibitor is yet available in therapy. We previously identified a series of 1,2,4-triazole-3-thione derivatives acting as micromolar inhibitors of MBLs in vitro, but devoid of synergistic activity in microbiological assays. Here, via a multidisciplinary approach, including molecular modelling, synthesis, enzymology, microbiology, and X-ray crystallography, we optimized this series of compounds and identified low micromolar inhibitors active against clinically relevant MBLs (NDM-1- and VIM-type). The best inhibitors increased, to a certain extent, the susceptibility of NDM-1- and VIM-4-producing clinical isolates to meropenem. X-ray structures of three selected inhibitors in complex with NDM-1 elucidated molecular recognition at the base of potency improvement, confirmed in silico predicted orientation, and will guide further development steps.

Vibrio cholerae causes life-threatening infections in low-income countries due to the rise of antibacterial resistance. Innovative pharmacological targets have been investigated and carbonic anhydrases (CAs, EC: 4.2.1.1) encoded by V. cholerae ( Vch CAs) emerged as a valuable option. Recently, we developed a large library of para - and meta -benzenesulfonamides characterised by moieties with a different flexibility degree as CAs inhibitors. Stopped flow-based enzymatic assays showed strong inhibition of Vch αCA for this library, while lower affinity was detected against the other isoforms. In particular, cyclic urea 9c emerged for a nanomolar inhibition of Vch αCA ( K I = 4.7 nM) and high selectivity with respect to human isoenzymes (SI≥ 90). Computational studies revealed the influence of moiety flexibility on inhibitory activity and isoform selectivity and allowed accurate SARs. However, although Vch CAs are involved in the bacterium virulence and not in its survival, we evaluated the antibacterial activity of such compounds, resulting in no direct activity.

The plethora of viral outbreaks experienced in the last decade, together with the widespread distribution of many re-emerging and newly emerging viruses, emphasize the urgent need for novel broad-spectrum antivirals as tools for early intervention in case of future epidemics. Non-natural nucleosides have been at the forefront for the treatment of infectious diseases for many years and still represent one of the most successful classes of antiviral molecules on the market. In the attempt to explore the biologically relevant chemical space of this class of antimicrobials, we describe herein the development of novel base-modified nucleosides by converting previously identified 2,6-diaminopurine antivirals into the corresponding D/L ribonucleosides, acyclic nucleosides and prodrug derivatives. A phenotypic screening against viruses belonging to different families (Flaviviridae, Coronaviridae, Retroviridae) and against a panel of Gram-positive and Gram-negative bacteria, allowed to identify a few interesting molecules with broad-spectrum antimicrobial activities., (© 2023 Wiley-VCH Verlag GmbH.)

Addressing antibacterial resistance is a major concern of the modern world. The development of new approaches to meet this deadly threat is a critical priority. In this article, we investigate a new approach to negate bacterial resistance: exploit the β-lactam bond cleavage by β-lactamases to selectively trigger antibacterial prodrugs into the bacterial periplasm. Indeed, multidrug-resistant Gram-negative pathogens commonly produce several β-lactamases that are able to inactivate β-lactam antibiotics, our most reliable and widely used therapeutic option. The chemical structure of these prodrugs is based on a monobactam promoiety, covalently attached to the active antibacterial substance, zidovudine (AZT). We describe the synthesis of 10 prodrug analogues ( 5a-h ) in four to nine steps and their biological activity. Selective enzymatic activation by a panel of β-lactamases is demonstrated, and subsequent structure-activity relationships are discussed. The best compounds are further evaluated for their activity on both laboratory strains and clinical isolates, preliminary stability, and toxicity.

Trifluoroacetic acid (TFA), due to its strong acidity and low boiling point, is extensively used in protecting groups-based synthetic strategies. Indeed, synthetic compounds bearing basic functions, such as amines or guanidines (commonly found in peptido or peptidomimetic derivatives), developed in the frame of drug discovery programmes, are often isolated as trifluoroacetate (TF-Acetate) salts and their biological activity is assessed as such in in vitro, ex vivo, or in vivo experiments. However, the presence of residual amounts of TFA was reported to potentially affect the accuracy and reproducibility of a broad range of cellular assays (e. g. antimicrobial susceptibility testing, and cytotoxicity assays) limiting the further development of these derivatives. Furthermore, the impact of the counterion on biological activity, including TF-Acetate, is still controversial. Herein, we present a focused case study aiming to evaluate the activity of an antibacterial AlkylGuanidino Urea (AGU) compound obtained as TF-Acetate (1a) and hydrochloride (1b) salt forms to highlight the role of counterions in affecting the biological activity. We also prepared and tested the corresponding free base (1c). The exchange of the counterions applied to polyguanidino compounds represents an unexplored and challenging field, which required significant efforts for the successful optimization of reliable methods of preparation, also reported in this work. In the end, the biological evaluation revealed a quite similar biological profile for the salt derivatives 1a and 1b and a lower potency was found for the free base 1c., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

New classes of antibacterial drugs are urgently needed to address the global issue of antibiotic resistance. In this context, peptaibols are promising membrane-active peptides since they are not involved in innate immunity and their antimicrobial activity does not involve specific cellular targets, therefore reducing the chance of bacterial resistance development. Trichogin GA IV is a nonhemolytic, natural, short-length peptaibol active against Gram-positive bacteria and resistant to proteolysis. In this work, we report on the antibacterial activity of cationic trichogin analogs. Several peptides appear non-hemolytic and strongly active against many clinically relevant bacterial species, including antibiotic-resistant clinical isolates, such as Staphylococcus aureus , Acinetobacter baumannii , and extensively drug-resistant Pseudomonas aeruginosa , against which there are only a limited number of antibiotics under development. Our results further highlight how the modification of natural peptides is a valuable strategy for obtaining improved antibacterial agents with potential therapeutic applications.

Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S . lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S . lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and 1 H/ 13 C NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.

Metallo-β-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 ( K i = 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.

The diffusion of antibiotic-resistant, Gram-negative, opportunistic pathogens, an increasingly important global public health issue, causes a significant socioeconomic burden. Acinetobacter baumannii isolates, despite causing a lower number of infections than Enterobacterales, often show multidrug-resistant phenotypes. Carbapenem resistance is also rather common, prompting the WHO to include carbapenem-resistant A. baumannii as a "critical priority" for the discovery and development of new antibacterial agents. In a previous work, we identified several series of compounds showing either direct-acting or synergistic activity against relevant Gram-negative species, including A. baumannii. Among these, two pyrazole compounds, despite being devoid of any direct-acting activity, showed remarkable synergistic activity in the presence of a subinhibitory concentration of colistin on K. pneumoniae and A. baumannii and served as a starting point for the synthesis of new analogues. In this work, a new series of 47 pyrazole compounds was synthesized. Some compounds showed significant direct-acting antibacterial activity on Gram-positive organisms. Furthermore, an evaluation of their activity as potential antibiotic adjuvants allowed for the identification of two highly active compounds on MDR Acinetobacter baumannii , including colistin-resistant isolates. This work confirms the interest in pyrazole amides as a starting point for the optimization of synergistic antibacterial compounds active on antibiotic-resistant, Gram-negative pathogens.

Metallo-β-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 β-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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cystic fibrosis (CF) is a genetic disease caused by loss-of-function mutations in the CFTR gene, which codes for a defective ion channel. This causes an electrolyte imbalance and results in a spiral of negative effects on multiple organs, most notably the accumulation of thick mucus in the lungs, chronic respiratory tract infections and inflammation leading to pulmonary exacerbation and premature death. Progressive decline of lung function is mainly linked to persistent or recurring infections, mostly caused by bacteria, which require treatments with antibiotics and represent one of the major life-limiting factors in subjects with CF. Treatment of such a complex disease require multiple drugs with a consequent therapeutic burden and complications caused by drug-drug interactions and rapid emergence of bacterial drug resistance. We report herein our recent efforts in developing innovative multifunctional antibiotics specifically tailored to CF by a direct action on bacterial topoisomerases and a potential indirect effect on the pulmonary mucociliary clearance mediated by ΔF508-CFTR correction. The obtained results may pave the way for the development of a simplified therapeutic approach with a single agent acting as multifunctional Antibacterial-Corrector., (© 2022 Wiley-VCH GmbH.)

Metallo-β-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 K i 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 β-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., (© 2022 Wiley-VCH GmbH.)

The ever-faster rise of antimicrobial resistance (AMR) represents a major global Public Health challenge. New chemical entities with innovative Modes of Action (MoAs) are thus desirable. We recently reported the development of a novel class of broad-spectrum bactericidal agents, the AlkylGuanidino Ureas (AGU). Due to their polycationic structure, they likely target bacterial membranes. In order to better understand their MoA, we synthesized a library of AGU derivatives by structural simplification of selected hit compounds and developed specific assays based on membrane models by means of both analytical and computational techniques. Cell-based assays provided experimental evidence that AGUs disrupt bacterial membranes without showing hemolytic behavior. Hence, we herein report a thorough chemical and biological characterization of a new series of AGUs obtained through molecular simplification, allowing the rational design of potent antibacterial compounds active on antibiotic-resistant strains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Metallo-β-lactamases (MBLs) are important contributors of Gram-negative bacteria resistance to β-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 K i 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

A protracted outbreak of New Delhi metallo-β-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the bla NDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-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-β-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 K i 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Resistance to β-lactam antibiotics in Gram-negatives producing metallo-β-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with K i values in the μM to sub-μM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the potentiation activity of the compounds was limited by poor outer membrane penetration or efflux. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E. coli clinical strain toward several β-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-β-lactamases (MBLs), which represent major resistance determinants to β-lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series); the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with K i values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of β-lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL.

The application of various isonitrile-based multicomponent reactions to protected (2-oxoethyl)boronic acid (as the carbonyl component) is described. The Ugi reaction, both in the four components and in the four centers-three components versions, and the van Leusen reaction, proved effective at providing small libraries of MIDA-protected β-aminoboronic acids. The corresponding free β-aminoboronic acids, quantitatively recovered through basic mild deprotection, were found to be quite stable and were fully characterized, including by 11 B-NMR spectroscopy. Single-crystal X-ray diffraction analysis, applied both to a MIDA-protected and a free β-aminoboronic acid derivative, provided evidence for different conformations in the solid-state. Finally, the antimicrobial activities of selected compounds were evaluated by measuring their minimal inhibitory concentration (MIC) values, and the binding mode of the most promising derivative on OXA-23 class D β-lactamase was predicted by a molecular modeling study., Competing Interests: The authors declare no conflict of interest

Antibiotic resistance is an increasingly important global public health issue, as major opportunistic pathogens are evolving toward multidrug- and pan-drug resistance phenotypes. New antibiotics are thus needed to maintain our ability to treat bacterial infections. According to the WHO, carbapenem-resistant Acinetobacter , Enterobactericaeae , and Pseudomonas are the most critical targets for the development of new antibacterial drugs. An automated phenotypic screen was implemented to screen 634 synthetic compounds obtained in-house for both their direct-acting and synergistic activity. Fourteen percent and 10% of the compounds showed growth inhibition against tested Gram-positive and Gram-negative bacteria, respectively. The most active direct-acting compounds showed a broad-spectrum antibacterial activity, including on some multidrug-resistant clinical isolates. In addition, 47 compounds were identified for their ability to potentiate the activity of other antibiotics. Compounds of three different scaffolds (2-quinolones, phenols, and pyrazoles) showed a strong potentiation of colistin, some being able to revert colistin resistance in Acinetobacter baumannii ., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Nowadays, the increasing of multidrug-resistant pathogenic bacteria represents a serious threat to public health, and the lack of new antibiotics is becoming a global emergency. Therefore, research in antibacterial fields is urgently needed to expand the currently available arsenal of drugs. We have recently reported an alkyl-guanidine derivative (2), characterized by a symmetrical dimeric structure, as a good candidate for further developments, with a high antibacterial activity against both Gram-positive and Gram-negative strains. In this study, starting from its chemical scaffold, we synthesized a small library of analogues. Moreover, biological and in vitro pharmacokinetic characterizations were conducted on some selected derivatives, revealing notable properties: broad-spectrum profile, activity against resistant clinical isolates, and appreciable aqueous solubility. Interestingly, 2 seems neither to select for resistant strains nor to macroscopically alter the membranes, but further studies are required to determine the mode of action.