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The direct photoexcitation of isocyanides is able to trigger a collection of Ugi-like multicomponent reactions under metal-free conditions and in the absence of any additives starting from both linear and cyclic tertiary aromatic amines.

The merging of micellar and photoredox catalysis represents a key issue to promote “in water” photochemical transformations. A photomicellar catalyzed synthesis of amides from N-methyl-N-alkyl aromatic amines and both aliphatic and aromatic isocyanides is herein presented. The mild reaction conditions enabled a wide substrate scope and a good functional groups tolerance, as further shown in the late-stage functionalization of complex bioactive scaffolds. Furthermore, solution 1D and 2D NMR experiments performed, for the first time, in the presence of paramagnetic probes enabled the study of the reaction environment at the atomic level along with the localization of the photocatalyst with respect to the micelles, thus providing experimental data to drive the identification of optimum photocatalyst/surfactant pairing.

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of “selectivity index” in comparison to 1.

SARS-CoV-2 infection is still spreading worldwide, and new antiviral therapies are an urgent need to complement the approved vaccine preparations. SARS-CoV-2 nps13 helicase is a validated drug target participating in the viral replication complex and possessing two associated activities: RNA unwinding and 5′-triphosphatase. In the search of SARS-CoV-2 direct antiviral agents, we established biochemical assays for both SARS-CoV-2 nps13-associated enzyme activities and screened both in silico and in vitro a small in-house library of natural compounds. Myricetin, quercetin, kaempferol, and flavanone were found to inhibit the SARS-CoV-2 nps13 unwinding activity at nanomolar concentrations, while licoflavone C was shown to block both SARS-CoV-2 nps13 activities at micromolar concentrations. Mode of action studies showed that all compounds are nsp13 noncompetitive inhibitors versus ATP, while computational studies suggested that they can bind both nucleotide and 5′-RNA nsp13 binding sites, with licoflavone C showing a unique pattern of interaction with nsp13 amino acid residues. Overall, we report for the first time natural flavonoids as selective inhibitors of SARS-CoV-2 nps13 helicase with low micromolar activity.

Background This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi. Methods R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux. Results R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460. Conclusions Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.

Antibiotic resistance breakers, such as efflux pump inhibitors (EPIs), represent a powerful alternative to the development of new antimicrobials. Recently, by using previously described EPIs, we developed pharmacophore models able to identify inhibitors of NorA, the most studied efflux pump of Staphylococcus aureus. Herein we report the pharmacophore‐based virtual screening of a library of new potential NorA EPIs generated by an in‐silico scaffold hopping approach of the quinoline core. After chemical synthesis and biological evaluation of the best virtual hits, we found the quinazoline core as the best performing scaffold. Accordingly, we designed and synthesized a series of functionalized 2‐arylquinazolines, which were further evaluated as NorA EPIs. Four of them exhibited a strong synergism with ciprofloxacin and a good inhibition of ethidium bromide efflux on resistant S. aureus strains coupled with low cytotoxicity against human cell lines, thus highlighting a promising safety profile., Resistance breakers: By using scaffolds of approved drugs, we performed a scaffold hopping of the quinoline core coupled with a pharmacophore‐based virtual screen. The best hits were synthesized and evaluated as NorA efflux pump inhibitors (EPIs), and two 2‐arylquinazoline analogues were identified as potent NorA EPIs nontoxic toward human cells.

The newly emerged coronavirus, called SARS-CoV-2, is the causing pathogen of pandemic COVID-19. The identification of drugs to treat COVID-19 and other coronavirus diseases is an urgent global need, thus different strategies targeting either virus or host cell are still under investigation. Direct-acting agents, targeting protease and polymerase functionalities, represent a milestone in antiviral therapy. The 3C-like (or Main) protease (3CLpro) and the nsp12 RNA-dependent RNA-polymerase (RdRp) are the best characterized SARS-CoV-2 targets and show the highest degree of conservation across coronaviruses fostering the identification of broad-spectrum inhibitors. Coronaviruses also possess a papain-like protease, another essential enzyme, still poorly characterized and not equally conserved, limiting the identification of broad-spectrum agents. Herein, we provide an exhaustive comparative analysis of SARS-CoV-2 proteases and RdRp with respect to other coronavirus homologues. Moreover, we highlight the most promising inhibitors of these proteins reported so far, including the possible strategies for their further development.

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 &micro, g/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones.

[Image: see text] Treatment of dengue virus (DENV) and other flavivirus infections is an unmet medical need. The highly conserved flaviviral NS5 RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target that interacts with NS3 and viral RNA within the replication complex assembly. Biochemical and cell-based evidence indicate that targeting cavity B may lead to dual RdRp and NS5–NS3 interaction inhibitors. By ligand-based design around 1H-pyrido[2,1-b][1,3]benzothiazol-1-one (PBTZ) 1, we identified new potent and selective DENV inhibitors that exert dual inhibition of NS5 RdRp and NS3–NS5 interaction, likely through binding cavity B. Resistance studies with compound 4 generated sequence variants in the 3′-untranslated region of RNA while further biochemical experiments demonstrated its ability to block also RNA-NS5 interaction, required for correct RNA synthesis in cells. These findings shed light on the potential mechanism of action for this class of compounds, underlying how PBTZs are very promising lead candidates for further evaluation.

Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA).

RNA dependent RNA polymerases (RdRp) are essential enzymes for flavivirus replication. Starting from an in silico docking analysis we identified a pyridobenzothiazole compound, HeE1-2Tyr, able to inhibit West Nile and Dengue RdRps activity in vitro, which proved effective against different flaviviruses in cell culture. Crystallographic data show that HeE1-2Tyr binds between the fingers domain and the priming loop of Dengue virus RdRp (Site 1). Conversely, enzyme kinetics, binding studies and mutational analyses suggest that, during the catalytic cycle and assembly of the RdRp-RNA complex, HeE1-2Tyr might be hosted in a distinct binding site (Site 2). RdRp mutational studies, driven by in silico docking analysis, allowed us to locate the inhibition Site 2 in the thumb domain. Taken together, our results provide innovative concepts for optimization of a new class of anti-flavivirus compounds.

Antimicrobial resistance (AMR) represents a hot topic in drug discovery. Besides the identification of new antibiotics, the use of nonantibiotic molecules to block resistance mechanisms is a powerful alternative. Bacterial efflux pumps exert an early step in AMR development by allowing bacteria to grow at subinhibitorial drug concentrations. Thus, efflux pump inhibitors (EPIs) offer a great opportunity to fight AMR. Given our experience in developing Staphylococcus aureus NorA EPIs, in this work, starting from the 2-phenylquinoline hit 1, we planned the introduction of methoxy groups on the basis of their presence in known NorA EPIs. Among the 35 different synthesized derivatives, compounds 3b and 7d exhibited the best NorA inhibition activity by restoring at very low concentrations ciprofloxacin MICs against resistant S. aureus strains. Interestingly, both compounds displayed EPI activities at nontoxic concentrations for human cells as well as highlighted promising results by preliminary pharmacokinetic studies.

Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone – PQQ4R), againstEscherichia coli,by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux inE. colireducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of theE. coliinner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future adjuvants of conventional therapy againstE. coliand other Gram-negative bacteria, especially their multidrug resistant forms. Their major limitation is the high toxicity for human cells at the concentrations needed to be effective against bacteria. Their future molecular optimization to improve the efflux inhibitory properties and reduce relative toxicity will optimize their potential for clinical usage against multi-drug resistant bacterial infections due to efflux.

We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl] quinolone derivative 8 proved to be the best compound of this series, exhibiting IC50 value of 0.069 µM against NS5B polymerase and selective antiviral effect (EC50 = 3.03 µM, EC90 =13.5 µM) coupled with the absence of any cytostatic effect (CC50 >163 µM, SI >54) in Huh-9-13 cells carrying a HCV genotype 1b, as measured by MTS assay. These results indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs.

Objectives A number of previous studies has provided evidence that the well-known anti-bacterial quinolones may have potential as anti-cancer drugs. The aim of this study was to evaluate potential anti-tumour activity and selectivity of a set of 6-aminoquinolones showing some chemical similarity to naphthyridone derivative CX-5461, recently described as innovative anti-cancer agent. Materials and methods In-house quinolones 1-8 and ad hoc synthesized derivatives 9-13 were tested on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and mesenchymal progenitor (MePR2B) cell lines, analysing their effects on the cell cycle and cell death using FACS methodology. Activation of p53 was evaluated by western blotting. Results Benzyl esters 4, 5 and their amide counterparts 12, 13 drastically modulated MCF-7 cell cycles inducing DNA fragmentation and cell death, thus proving to be potential anti-tumour compounds. When assayed in non-tumour MePR2B cells, compounds 4 and 5 were cytotoxic while 12 and 13 had a certain degree of selectivity, with compound 12 emerging as the most promising. Western blot analysis revealed that severe p53-K382ac activation was promoted by benzylester 5. In contrast, amide 12 exerted only a moderate effect which was, however, comparable to that of suberoylanilide hydoxamic acid (SAHA). Conclusions Taken together, these results further reinforce evidence that quinolones have potential as anti-cancer agents. Future work will be focused on understanding compound 12 mechanisms of action, and to obtain more potent and selective compounds.

We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counter screen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting IC50 value of 7.9 µM against NS5B polymerase and antiviral effect (EC50 = 8.1 µM, EC90 = 23.3 µM) coupled with the absence of any antimetabolic effect (CC50 >224 µM, SI >28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment.

The pandemic evolution of SARS-CoV-2 infection is forcing the scientific community to unprecedented efforts to explore all possible approaches against COVID-19. In this context, targeting virus entry is a promising antiviral strategy for controlling viral infections. The main strategies pursued to inhibit the viral entry are considering both the virus and the host factors involved in the process. Primarily, direct-acting antivirals rely on inhibition of the interaction between ACE2 and the receptor binding domain (RBD) of the Spike (S) protein or targeting the more conserved heptad repeats (HRs), involved in the membrane fusion process. The inhibition of host TMPRSS2 and cathepsins B/L may represent a complementary strategy to be investigated. In this review, we discuss the development entry inhibitors targeting the S protein, as well as the most promising host targeting strategies involving TMPRSS2 and CatB/L, which have been exploited so far against CoVs and other related viruses.