The discovery of aryl-2-nitroethyl triamino pyrimidines as anti-Trypanosoma brucei agents.

Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-ß-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.

Structure-Based Optimization of 1,2,4-Triazole-3-Thione Derivatives: Improving Inhibition of NDM-/VIM-Type Metallo-ß-Lactamases and Synergistic Activity on Resistant Bacteria.

The worldwide emergence and dissemination of Gram-negative bacteria expressing metallo-ß-lactamases (MBLs) menace the efficacy of all ß-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.

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.

Novel Targets and Mechanisms in Antimicrobial Drug Discovery.

The spread of infections resistant to available anti-infective drugs is a serious menace to human health [...].

Can We Exploit ß-Lactamases Intrinsic Dynamics for Designing More Effective Inhibitors?

ß-lactamases (BLs) represent the most frequent cause of antimicrobial resistance in Gram-negative bacteria. Despite the continuous efforts in the development of BL inhibitors (BLIs), new BLs able to hydrolyze the last developed antibiotics rapidly emerge. Moreover, the insurgence rate of effective mutations is far higher than the release of BLIs able to counteract them. This results in a shortage of antibiotics that is menacing the effective treating of infectious diseases. The situation is made even worse by the co-expression in bacteria of BLs with different mechanisms and hydrolysis spectra, and by the lack of inhibitors able to hit them all. Differently from other targets, BL flexibility has not been deeply exploited for drug design, possibly because of the small protein size, for their apparent rigidity and their high fold conservation. In this mini-review, we discuss the evidence for BL binding site dynamics being crucial for catalytic efficiency, mutation effect, and for the design of new inhibitors. Then, we report on identified allosteric sites in BLs and on possible allosteric inhibitors, as a strategy to overcome the frequent occurrence of mutations in BLs and the difficulty of competing efficaciously with substrates. Nevertheless, allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility towards available antibiotics.

Virtual screening identifies broad-spectrum ß-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases.

Bacteria are known to evade ß-lactam antibiotic action by producing ß-lactamases (BLs), including carbapenemases, which are able to hydrolyze nearly all available ß-lactams. The production of BLs represents one of the best known and most targeted mechanisms of resistance in bacteria. We have performed the parallel screening of commercially available compounds against a panel of clinically relevant BLs: class A CTX-M-15 and KPC-2, subclass B1 NDM-1 and VIM-2 MBLs, and the class C P. aeruginosa AmpC. The results show that all BLs prefer scaffolds having electron pair donors: KPC-2 is preferentially inhibited by sulfonamide and tetrazole-based derivatives, NDM-1 by compounds bearing a thiol, a thiosemicarbazide or thiosemicarbazone moiety, while VIM-2 by triazole-containing molecules. Few broad-spectrum BLs inhibitors were identified; among these, compound 40 potentiates imipenem activity against an NDM-1-producing E. coli clinical strain. The binary complexes of the two most promising compounds binding NDM-1 and VIM-2 were obtained at high resolution, providing strong insights to improve molecular docking simulations, especially regarding the interaction of MBLs with inhibitors.

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.

Ten Years with New Delhi Metallo-ß-lactamase-1 (NDM-1): From Structural Insights to Inhibitor Design.

The worldwide emergence of New Delhi metallo-ß-lactamase-1 (NDM-1) as a carbapenemase able to hydrolyze nearly all available ß-lactam antibiotics has characterized the past decade, endangering efficacious antibacterial treatments. No inhibitors for NDM-1 are available in therapy, nor are promising compounds in the pipeline for future NDM-1 inhibitors. We report the studies dedicated to the design and development of effective NDM-1 inhibitors. The discussion for each agent moves from the employed design strategy to the ability of the identified inhibitor to synergize ß-lactam antibiotics. A structural analysis of NDM-1 mechanism of action based on selected X-ray complexes is also reported: the intrinsic flexibility of the binding site and the comparison between penicillin/cephalosporin and carbapenem mechanisms of hydrolysis are evaluated. Despite the valuable progress in terms of structural and mechanistic information, the design of a potent NDM-1 inhibitor to be introduced in therapy remains challenging. Certainly, only the deep knowledge of NDM-1 architecture and of the variable mechanism of action that NDM-1 employs against different classes of substrates could orient a successful drug discovery campaign.

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.

Conformational Propensity and Biological Studies of Proline Mutated LR Peptides Inhibiting Human Thymidylate Synthase and Ovarian Cancer Cell Growth.

LR and [d-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pro n]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.

Structure-Based Virtual Screening for the Discovery of Novel Inhibitors of New Delhi Metallo-ß-lactamase-1.

Bacterial resistance has become a worldwide concern after the emergence of metallo-ß-lactamases (MBLs). They represent one of the major mechanisms of bacterial resistance against beta-lactam antibiotics. Among MBLs, New Delhi metallo-ß-lactamase-1 NDM-1, the most prevalent type, is extremely efficient in inactivating nearly all-available antibiotics including last resort carbapenems. No inhibitors for NDM-1 are currently available in therapy, making the spread of NDM-1 producing bacterial strains a serious menace. With this perspective, we performed a structure-based in silico screening of a commercially available library using FLAPdock and identified several, non-ß-lactam derivatives as promising candidates active against NDM-1. The binding affinities of the highest scoring hits were measured in vitro revealing, for some of them, low micromolar affinity toward NDM-1. For the best inhibitors, efficacy against resistant bacterial strains overexpressing NDM-1 was validated, confirming their favorable synergistic effect in combination with the carbapenem Meropenem.

Phenylboronic Acid Derivatives as Validated Leads Active in Clinical Strains Overexpressing KPC-2: A Step against Bacterial Resistance.

The emergence and dissemination of multidrug resistant (MDR) pathogens resistant to nearly all available antibiotics poses a significant threat in clinical therapy. Among them, Klebsiella pneumoniae clinical isolates overexpressing KPC-2 carbapenemase are the most worrisome, extending bacterial resistance to last-resort carbapenems. In this study, we investigate the molecular recognition requirements in the KPC-2 active site by small phenylboronic acid derivatives. Four new phenylboronic acid derivatives were designed and tested against KPC-2. For the most active, despite their simple chemical structures, nanomolar affinity was achieved. The new derivatives restored susceptibility to meropenem in clinical strains overexpressing KPC-2. Moreover, no cytotoxicity was detected in cell-viability assays, which further validated the designed leads. Two crystallographic binary complexes of the best inhibitors binding KPC-2 were obtained at high resolution. Kinetic descriptions of slow binding, time-dependent inhibition, and interaction geometries in KPC-2 were fully investigated. This study will ultimately lead toward the optimization and development of more-effective KPC-2 inhibitors.

Computational and biological profile of boronic acids for the detection of bacterial serine- and metallo-ß-lactamases.

ß-Lactamases (BLs) able to hydrolyze ß-lactam antibiotics and more importantly the last resort carbapenems, represent a major mechanism of resistance in Gram-negative bacteria showing multi-drug or extensively drug resistant phenotypes. The early detection of BLs responsible of resistant infections is challenging: approaches aiming at the identification of new BLs inhibitors (BLI) can thus serve as the basis for the development of highly needed diagnostic tools. Starting from benzo-[b]-thiophene-2-boronic acid (BZB), a nanomolar inhibitor of AmpC ß-lactamase (K i = 27 nM), we have identified and characterized a set of BZB analogues able to inhibit clinically-relevant ß-lactamases, including AmpC, Extended-Spectrum BLs (ESBL), KPC- and OXA-type carbapenemases and metallo-ß-lactamases (MBL). A multiligand set of boronic acid (BA) ß-lactamase inhibitors was obtained using covalent molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. Data confirmed the possibility to discriminate between clinically-relevant ß-lactamases on the basis of their inhibition profile. Interestingly, this work also allowed the identification of potent KPC-2 and NDM-1 inhibitors able to potentiate the activity of cefotaxime (CTX) and ceftazidime (CAZ) against resistant clinical isolates (MIC reduction, 32-fold). Our results open the way to the potential use of our set of compounds as a diagnostic tool for the sensitive detection of clinically-relevant ß-lactamases.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

BACKGROUND: RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. PURPOSE: Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. METHODS: The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. RESULTS: Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. CONCLUSION: In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity.