Salvianolic Acid B Strikes Back: New Evidence in the Modulation of Expression and Activity of Matrix Metalloproteinase 9 in MDA-MB-231 Human Breast Cancer Cells.

Salvianolic acid B (SalB) is a bioactive compound from Salviae miltiorrhizae, one of the most important traditional herbal medicines widely used in several countries for the treatment of cardiovascular diseases. The aim of this study was to evaluate the in vitro effect of SalB on the expression and the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent proteolytic enzyme, in human MDA-MB-231 breast cancer cells. This cellular model is characterized by a marked invasive phenotype, supported by a high constitutive expression of MMPs, especially gelatinases. SalB was first of all evaluated by in silico approaches primarily aimed at predicting the main pharmacokinetic parameters. The most favorable interaction between the natural compound and MMP-9 was instead tested by molecular docking analysis that was subsequently verified by an enzymatic inhibition assay. MDA-MB-231 cells were treated with SalB 5 µM and 50 µM for 24 h and 48 h. The conditioned media obtained from treated cells were then analyzed by gelatin zymography and reverse zymography to, respectively, evaluate the MMP-9 activity and the presence of TIMP-1. The expression of the enzyme was then evaluated by Western blot on conditioned media and by analysis of transcripts through reverse transcriptase-polymerase chain reaction (RT-PCR). The in silico approach showed the ability of SalB to interact with the catalytic zinc ion of the enzyme, with a plausible competitive mode of action. The analysis of conditioned culture media showed a reduction in MMP-9 activity and the concomitant decrease in the enzyme concentration, partially confirmed by analysis of transcripts. SalB showed the ability to modulate the function of MMP-9 in MDA-MB-231 cells. To our knowledge, this is the first time in which the role of SalB on MMP-9 in a highly invasive cellular model is investigated. The obtained results impose further and more specific evaluations in order to obtain a better understanding of the biochemical mechanisms that regulate the interaction between this natural compound and the MMP-9.

Nanobodies targeting LexA autocleavage disclose a novel suppression strategy of SOS-response pathway.

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections.

Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators.

The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CLpro. The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CLpro. The compounds are slow-binding inactivators of 3CLpro with a Ki of 3.95 µM and 3.77 µM for protocetraric and salazinic acid, respectively, and inhibitory efficiency kinact/Ki at about 3 × 10-5 s-1µM-1. The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 µM, which is well below the Ki values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CLpro. The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors.

Quatsomes Formulated with l-Prolinol-Derived Surfactants as Antibacterial Nanocarriers of (+)-Usnic Acid with Antioxidant Activity.

The efficacy of the treatment of bacterial infection is seriously reduced because of antibiotic resistance; thus, therapeutic solutions against drug-resistant microbes are necessary. Nanoparticle-based solutions are particularly promising for meeting this challenge because they can offer intrinsic antimicrobial activity and sustained drug release at the target site. Herein, we present a newly developed nanovesicle system of the quatsome family, composed of l-prolinol-derived surfactants and cholesterol, which has noticeable antibacterial activity even on Gram-negative strains, demonstrating great potential for the treatment of bacterial infections. We optimized the vesicle stability and antibacterial activity by tuning the surfactant chain length and headgroup charge (cationic or zwitterionic) and show that these quatsomes can furthermore serve as nanocarriers of pharmaceutical actives, demonstrated here by the encapsulation of (+)-usnic acid, a natural substance with many pharmacological properties.

Structurally Related Liposomes Containing N-Oxide Surfactants: Physicochemical Properties and Evaluation of Antimicrobial Activity in Combination with Therapeutically Available Antibiotics.

Although liposomes are largely investigated as drug delivery systems, they can also exert a pharmacological activity if devoid of an active principle as a function of their composition. Specifically, charged liposomes can electrostatically interact with bacterial cells and, in some cases, induce bacterial cell death. Moreover, they also show a high affinity toward bacterial biofilms. We investigated the physicochemical and antimicrobial properties of liposomes formulated with a natural phospholipid and four synthetic l-prolinol-derived surfactants at 9/1 and 8/2 molar ratios. The synthetic components differ in the nature of the polar headgroup (quaternary ammonium salt or N-oxide) and/or the length of the alkyl chain (14 or 16 methylenes). These differences allowed us to investigate the effect of the molecular structure of liposome components on the properties of the aggregates and their ability to interact with bacterial cells. The antimicrobial properties of the different formulations were assessed against Gram-negative and Gram-positive bacteria and fungi. Drug-drug interactions with four classes of available clinical antibiotics were evaluated against Staphylococcus spp. The target of each class of antibiotics plays a pivotal role in exerting a synergistic effect. Our results highlight that the liposomal formulations with an N-oxide moiety are required for the antibacterial activity against Gram-positive bacteria. In particular, we observed a synergism between oxacillin and liposomes containing 20 molar percentage of N-oxide surfactants onStaphylococcus haemolyticus, Staphylococcus epidermidis, andStaphylococcus aureus. In the case of liposomes containing 20 molar percentage of the N-oxide surfactant with 14 carbon atoms in the alkyl chain for S. epidermidis, the minimum inhibitory concentration was 0.125 µg/mL, well below the breakpoint value of the antibiotic.

Evaluation of the Analytical Performances of the Biolabo SOLEA 100 Optical Coagulometer and Comparison with the Stago STA-R MAX Analyser in the Determination of PT, APTT, and Fibrinogen.

INTRODUCTION: The Biolabo Solea 100 is a fully automated coagulation analyser using an optical system to detect coagulation designed to meet the needs of small- and medium-sized laboratories. This study aimed to evaluate the analytical performance in terms of bias, precision, and interference of the Biolabo Solea 100 coagulometer under routine laboratory conditions. In addition, a comparison was made with Stago STA-R MAX. MATERIALS AND METHODS: Imprecision and bias were evaluated for activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time (PT) at the medical decision levels. The results of 200, 181, and 206 plasma samples for APTT, FIB, and PT, respectively, were compared with those obtained by Stago STA-R MAX. In addition, the interference level of bilirubin, haemoglobin, triglycerides, and fractionated heparin was evaluated. RESULTS: Repeatability, intermediate imprecision, bias, and total error are overall below the defined limits of acceptability. Of interest is the high degree of agreement between Solea 100 and STA-R MAX with respect to PT (s), which fits perfectly with the theoretical line of identity (y = 0 + 1.00x). No interferences were found within the limits stated by the manufacturer, with some exceptions for APTT with heparin and APTT and PT for higher bilirubin concentrations. CONCLUSIONS: In conclusion, the performance of the Solea 100 optical analyser is satisfactory and adequate for the determination of routine coagulation tests. Moreover, they are perfectly comparable to mechanical systems, such as STA-R MAX and other upper-level analysers, even considering the low interference levels under routine conditions.

New and simplified method for drug combination studies by checkerboard assay.

To evaluate the effect of two combined antimicrobial drugs, one method currently in use is the checkerboard assay in a 96-well microplate, which gives a good in vivo estimation of the drug-drug combination effect. Appropriate and consolidated methods are described in numerous scientific publications which are, however, in turn, laborious and time-spending, specifically for the setting of the 96-well microplate preparation. Each drug of every combination must be prepared and dispensed individually in several steps, often limiting its use in terms of consumed materials and working time. In our method, the strengths of the previous consolidated techniques are kept, although the toughness and the execution time are drastically reduced. No special laboratory apparatuses are needed. All the procedures of our method can be referred to the CLSI or EUCAST guideline. The method provides few main steps, which can be summarised in:•Preparation of the microorganism inoculum and three concentrations of antimicrobial drugs.•Easy dispensing of all reagents into the microplates with a multichannel pipette.•Evaluation of the microorganism optical density (OD) by a microplate reader, and calculation of growth percentage for each of the 77 combinations.

Cyclic and Acyclic Amine Oxide Alkyl Derivatives as Potential Adjuvants in Antimicrobial Chemotherapy against Methicillin-Resistant Staphylococcus aureus with an MDR Profile.

The dramatic intensification of antimicrobial resistance occurrence in pathogenic bacteria concerns the global community. The revitalisation of inactive antibiotics is, at present, the only way to go through this health system crisis and the use of antimicrobial adjuvants is turning out the most promising approach. Due to their low toxicity, eco-friendly characteristics and antimicrobial activity, amphoteric surfactants are good candidates. This study investigated the adjuvant potentialities of commercial acyclic and newly cyclic N-oxide surfactants combined with therapeutically available antibiotics against MDR methicillin-resistant Staphylococcus aureus (MRSA). The safety profile of the new cyclic compounds, compared to commercial surfactants, was preliminarily assessed, evaluating the cytotoxicity on human peripheral mononuclear blood cells and the haemolysis in human red blood cells. The compounds show an efficacious antimicrobial activity strongly related to the length of the carbon atom chain. In drug-drug interaction assays, all surfactants act synergistically, restoring sensitivity to oxacillin in MRSA, with dodecyl acyclic and cyclic derivatives being the most effective. After evaluating the cytotoxicity and considering the antimicrobial action, the most promising compound is the L-prolinol amine-oxide C12NOX. These findings suggest that the combination of antibiotics with amphoteric surfactants is a valuable therapeutic option for topical infections sustained by multidrug-resistant S. aureus.

Transient disappearance of CD19+/CD5+ B-lymphocyte clone in peripheral blood in a patient with CLL during SARS-CoV-2-related mild disease.

Although lymphopenia is currently considered a good predictor for the prognosis of COVID-19, it must be critically evaluated in patients with CLL, where other clinical markers should be considered to define the prognosis and treatment.

Lactobacillus sakei Pro-Bio65 Reduces TNF-α Expression and Upregulates GSH Content and Antioxidant Enzymatic Activities in Human Conjunctival Cells.

Purpose: The study investigates the regulatory effects exhibited by lysate of Lactobacillus sakei pro-Bio65 (4%; L.SK) on the human conjunctival epithelial (HCE) cell line. Methods: Trypan blue and methylthiazol tetrazolium (MTT) methods were used to assess cell growth and viability. Mitochondrial membrane potential was assessed by JC-1 staining and cytofluorimetric detection methods. The antioxidant pattern and the intracellular reactive oxygen species (ROS) levels were analyzed by spectrophotometric and spectrofluorimetric methods. NF-κB luciferase activity was quantified by luminometric detection. NF-κB nuclear translocation, as well as mitochondrial morphology, were investigated by immunofluorescence using confocal microscopy. Cytokines and COX2 expression levels were determined by Western blot analyses. Results: This study demonstrates that L.SK exposure does not influence HCE cell proliferation and viability in vitro. L.SK paraprobiotic induces mild-low levels of intracellular ROS. It is coupled to changes in the mitochondrial membrane potential (ΔΨm), in a context of a regular mitochondrial-network organization. The negative modulation of tumor necrosis factor alpha (TNF-α) expression levels and rising antioxidant defense efficiency, mediated by the upregulation of glutathione (GSH) and increased antioxidant enzymatic activities, were observed. Conclusions: This study demonstrates that L.SK empowers the antioxidant endogenous efficiency of HCE cells, by the upregulation of the GSH content and the enzymatic antioxidant pattern, and concurrently reduces TNF-α protein expression. Translational Relevance: Although the obtained in vitro results should be confirmed by in vivo investigations, our data suggest the possibility of L.SK paraprobiotic application for promoting eye health, exploring its use as an endogen antioxidant system inducer in preventing and treating different oxidative stress-based, inflammatory, and age-related conditions.

The central role of the SOS DNA repair system in antibiotics resistance: A new target for a new infectious treatment strategy.

Bacteria have a considerable ability and potential to acquire resistance against antimicrobial agents by acting diverse mechanisms such as target modification or overexpression, multidrug transporter systems, and acquisition of drug hydrolyzing enzymes. Studying the mechanisms of bacterial cell physiology is mandatory for the development of novel strategies to control the antimicrobial resistance phenomenon, as well as for the control of infections in clinics. The SOS response is a cellular DNA repair mechanism that has an essential role in the bacterial biologic process involved in resistance to antibiotics. The activation of the SOS network increases the resistance and tolerance of bacteria to stress and, as a consequence, to antimicrobial agents. Therefore, SOS can be an applicable target for the discovery of new antimicrobial drugs. In the present review, we focus on the central role of SOS response in bacterial resistance mechanisms and its potential as a new target for control of resistant pathogens.

Antimycotic Activity of Ozonized Oil in Liposome Eye Drops against Candida spp.

Purpose: This study aims to investigate the antifungal activity and mechanism of action of ozonized oil eye drops in liposomes (Ozodrop), commercialized as eye lubricant for the treatment of dry eye syndrome and eye inflammation. The activity was tested against four clinical Candida species: Calbicans,Cglabrata,Ckrusei, and Corthopsilosis. Methods: The antifungal activity of the eye drop solution was ascertained by microdilution method in accordance with EUCAST obtaining the minimum inhibitory concentration for Ozodrop. The mechanism of action was further investigated in Calbicans by measuring cell vitality, intracellular reactive oxygen species production, levels of cellular and mitochondrial (∆Ψm) membrane potential, and the extent of membrane lipid peroxidation. Results: All Candida isolates were susceptible to Ozodrop with minimum inhibitory concentration values ranging from 0.195% (v/v) for Cglabrata to 6.25% (v/v) for Corthopsilosis. After 1 hour of exposure at the minimum inhibitory concentration value about 30% of cells were killed, reaching about 70% at the highest Ozodrop value. After Ozodrop exposure, Calbicans showed cell membrane depolarization, increased levels of lipid peroxidation, depolarized ∆Ψm, and increased reactive oxygen species generation. Conclusions: The significant increases in reactive oxygen species production cause the accumulation of reactive oxygen species-associated damages leading to progressive Candida cell dysfunction. Translational Relevance: The antifungal activity of Ozodrop was demonstrated at concentrations several times lower than the concentration that can be retrieved in ocular surface after its application. The antifungal activity of the eye drops Ozodrop would represent an interesting off-label indication for a product basically conceived as an eye lubricant.

Correlation of Physicochemical and Antimicrobial Properties of Liposomes Loaded with (+)-Usnic Acid.

(+)-Usnic acid (UA) is a natural substance that displays pharmacological activity, but it is barely soluble in water, so it was included in liposomes in order to study its properties. First, the effects of phospholipid structure and loading methodology on UA entrapment efficacy were evaluated. Then, the physicochemical and biological properties (UA delivery efficacy to Staphylococcus aureus bacterial cells) of different liposome formulations containing structurally related amphiphiles derived from L-prolinol were fully investigated. Entrapment efficiency of UA with passive loading by incubation was 80-100 molar percentage, which is related to lipophilicity of the drug and to the packing and fluidity of the bilayer. Some of the investigated formulations show the potential of UA in delivery systems (minimum inhibitory concentration of liposomal UA: 8 µg/mL) and even subtle variations of the molecular structure of lipids can significantly affect the liposomes' physicochemical properties and efficiency of drug release.

4-Amino-1,2,4-triazole-3-thione as a Promising Scaffold for the Inhibition of Serine and Metallo-ß-Lactamases.

The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available ß-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of ß-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine ß-Lactamase (SBLs) KPC-2 and class B1 metallo ß-Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.

Targeting the Class A Carbapenemase GES-5 via Virtual Screening.

The worldwide spread of ß-lactamases able to hydrolyze last resort carbapenems contributes to the antibiotic resistance problem and menaces the successful antimicrobial treatment of clinically relevant pathogens. Class A carbapenemases include members of the KPC and GES families. While drugs against KPC-type carbapenemases have recently been approved, for GES-type enzymes, no inhibitors have yet been introduced in therapy. Thus, GES carbapenemases represent important drug targets. Here, we present an in silico screening against the most prevalent GES carbapenemase, GES-5, using a lead-like compound library of commercially available compounds. The most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5 leading to four derivatives active as high micromolar competitive inhibitors. For the best inhibitors, the ability to inhibit KPC-2 was also evaluated. The discovered inhibitors constitute promising starting points for hit to lead optimization.

Inhibition of the transcriptional repressor LexA: Withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials.

AIMS: LexA protein is a transcriptional repressor which regulates the expression of more than 60 genes belonging to the SOS global regulatory network activated by damages to bacterial DNA. Considering its role in bacteria, LexA represents a key target to counteract bacterial resistance: the possibility to modulate SOS response through the inhibition of LexA autoproteolysis may lead to reduced drug susceptibility and acquisition of resistance in bacteria. In our study we investigated boron-containing compounds as potential inhibitors of LexA self-cleavage. MAIN METHODS: The inhibition of LexA self-cleavage was evaluated by following the variation of the first-order rate constant by LC-MS at several concentrations of inhibitors. In silico analysis was applied to predict the binding orientations assumed by the inhibitors in the protein active site, upon covalent binding to the catalytic Ser-119. Bacterial filamentation assay was used to confirm the ability of (3-aminophenyl)boronic acid to interfere with SOS induced activation. KEY FINDINGS: Boron-containing compounds act as inhibitors of LexA self-cleavage, as also confirmed by molecular modelling where the compounds interact with the catalytic Ser-119, via the formation of an acyl-enzyme intermediate. A new equation for the description of the inhibition potency in an autoproteolytic enzyme is also disclosed. Bacterial filamentation assays strongly support the interference of our compounds with the SOS response activation through inhibition of septum formation. SIGNIFICANCE: The obtained results demonstrated that phenylboronic compounds could be exploited in a hit-to-lead optimization process toward effective LexA self-cleavage inhibitors. They would sustain the rehabilitation in therapy of several dismissed antibiotics.

Phenylboronic Acids Probing Molecular Recognition against Class A and Class C ß-lactamases.

Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, ß-lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine ß-Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect ß-lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with ß-lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors.

X-ray Crystallography Deciphers the Activity of Broad-Spectrum Boronic Acid ß-Lactamase Inhibitors.

Recent decades have witnessed a dramatic increase of multidrug resistant (MDR) bacteria, compromising the efficacy of available antibiotics, and a continual decline in the discovery of novel antibacterials. We recently reported the first library of benzo[b]thiophen-2-ylboronic acid inhibitors sharing broad spectrum activity against ß-lactamases (BLs). The ability of these compounds to inhibit structurally and mechanistically different types of ß-lactamases has been here structurally investigated. An extensive X-ray crystallographic analysis of boronic acids (BAs) binding to proteins representative of serine BLs (SBLs) and metallo ß-lactamases (MBLs) have been conducted to depict the role played by the boronic group in driving molecular recognition, especially in the interaction with MBLs. Our derivatives are the first case of noncyclic boronic acids active against MBLs and represent a productive route toward potent broad-spectrum inhibitors.

First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase.

The worldwide spread of beta-lactamases with hydrolytic activity extended to last resort carbapenems is aggravating the antibiotic resistance problem and endangers the successful antimicrobial treatment of clinically relevant pathogens. As recently highlighted by the World Health Organization, new strategies to contain antimicrobial resistance are urgently needed. Class A carbapenemases include members of the KPC, GES and SFC families. These enzymes have the ability to hydrolyse penicillins, cephalosporins and carbapenems, while also being less susceptible to available beta-lactam inhibitors, such as clavulanic acid. The KPC family is the most prevalent. It is mostly found on plasmids in Klebsiella pneumoniae, meaning that great amounts of attention, in terms of inhibitor design and structural biology, have been dedicated to it, whereas no efforts have yet been dedicated to GES-type enzymes, despite their ability to rapidly and horizontally disseminate. We herein report the first in silico screening against GES-5, which is the most dangerous GES-type beta-lactamase, using a library of 800K commercially available candidates that all share drug-like properties, such as their MW, logP, rotatable bonds and HBA/HBD atoms. The best screening results were filtered to enrich the number of different chemotypes, and then submitted to molecular docking. The 34 most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5. Six hits acted as inhibitors, in the high micromolar range, towards GES-5 and led to the identification of the first, novel chemotypes with inhibitory activity against this clinically relevant carbapenemase.

In silico identification and experimental validation of hits active against KPC-2 ß-lactamase.

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type ß-lactamases are often reported as resistant to available ß-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non ß-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-ß-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the ß-lactam antibiotic meropenem by four-fold.