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Influenza viruses (IV) are single-stranded RNA viruses with a negative-sense genome and have the potential to cause pandemics. While vaccines exist for influenza, their protection is only partial. Additionally, there is only a limited number of approved anti-IV drugs, which are associated to emergence of drug resistance. To address these issues, for years we have focused on the development of small-molecules that can interfere with the heterodimerization of PA and PB1 subunits of the IV RNA-dependent RNA polymerase (RdRP). In this study, starting from a cycloheptathiophene-3-carboxamide compound that we recently identified, we performed iterative cycles of medicinal chemistry optimization that led to the identification of compounds 43 and 45 with activity in the nanomolar range against circulating A and B strains of IV. Mechanistic studies demonstrated the ability of 43 and 45 to interfere with viral RdRP activity by disrupting PA-PB1 subunits heterodimerization and to bind to the PA C-terminal domain through biophysical assays. Most important, ADME studies of 45 also showed an improvement in the pharmacokinetic profile with respect to the starting hit.
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Respiratory syncytial virus (RSV) stands as the foremost cause of infant hospitalization globally, ranking second only to malaria in terms of infant mortality. Although three vaccines have recently been approved for the prophylaxis of adults aged 60 and above, and pregnant women, there is currently no effective antiviral drug for treating RSV infections. The only preventive measure for infants at high risk of severe RSV disease is passive immunization through monoclonal antibodies. This Perspective offers an overview of the latest advancements in RSV drug discovery of small molecule antivirals, with particular focus on the promising findings from agents targeting the fusion and polymerase proteins. A comprehensive reflection on the current state of RSV research is also given, drawing inspiration from the lessons gleaned from HCV and HIV, while also considering the impact of the recent approval of the three vaccines.
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Antivirais , Descoberta de Drogas , Infecções por Vírus Respiratório Sincicial , Antivirais/farmacologia , Antivirais/química , Antivirais/uso terapêutico , Humanos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacosRESUMO
Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc2 155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance.
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Among the eight different triazolopyrimidine isomers existing in nature, 1,2,4-triazolo[1,5-a]pyrimidine (TZP) is one of the most studied and used isomers in medicinal chemistry. For some years, our group has been involved in developing regioselective one-pot procedures for the synthesis of 2-amino-7-aryl-5-methyl- and 2-amino-5-aryl-7-methyl-TZPs of interest in the preparation of antiviral agents. In this work, taking advantage of a Biginelli-like multicomponent reaction (MCR), we report the identification of finely tunable conditions to regioselectively synthesize C-6 ester-substituted amino-TZP analogues, both in dihydro and oxidized forms. Indeed, the use of mild acidic conditions is strongly directed toward the regioselective synthesis of 5-aryl-7-methyl C-6-substituted TZP analogues, while the use of neutral ionic liquids shifted the regioselectivity towards 7-aryl-5-methyl derivatives. In addition, the novel synthesized scaffolds were functionalized at the C-2 position and evaluated for their antiviral activity against RNA viruses (influenza virus, flaviviruses, and SARS-CoV-2). Compounds 25 and 26 emerged as promising anti-flavivirus agents, showing activity in the low micromolar range.
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Líquidos Iônicos , Isomerismo , Pirimidinas/química , EstereoisomerismoRESUMO
The superbug Staphylococcus aureus (S. aureus) exhibits several resistance mechanisms, including efflux pumps, that strongly contribute to antimicrobial resistance. In particular, the NorA efflux pump activity is associated with S. aureus resistance to fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from cells. Thus, since efflux pump inhibitors (EPIs) are able to increase antibiotic concentrations in bacteria as well as restore their susceptibility to these agents, they represent a promising strategy to counteract bacterial resistance. Additionally, the very recent release of two NorA efflux pump cryo-electron microscopy (cryo-EM) structures in complex with synthetic antigen-binding fragments (Fabs) represents a real breakthrough in the study of S. aureus antibiotic resistance. In this scenario, supervised molecular dynamics (SuMD) and molecular docking experiments were combined to investigate for the first time the molecular mechanisms driving the interaction between NorA and efflux pump inhibitors (EPIs), with the ultimate goal of elucidating how the NorA efflux pump recognizes its inhibitors. The findings provide insights into the dynamic NorA-EPI intermolecular interactions and lay the groundwork for future drug discovery efforts aimed at the identification of novel molecules to fight antimicrobial resistance.
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Antibacterianos , Infecções Estafilocócicas , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Staphylococcus aureus , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Microscopia Crioeletrônica , Farmacorresistência Bacteriana , Ciprofloxacina/farmacologia , Infecções Estafilocócicas/microbiologia , Proteínas de Bactérias/química , Testes de Sensibilidade MicrobianaRESUMO
One promising approach in treating antibiotic-resistant bacteria is to "break" resistances connected with antibacterial efflux by co-administering efflux pump inhibitors (EPIs) with antibiotics. Here, ten compounds, previously optimized to restore the susceptibility to ciprofloxacin (CIP) of norA-overexpressing Staphylococcus aureus, were evaluated for their ability to inhibit norA-mediated efflux in Staphylococcus pseudintermedius and synergize with CIP, ethidium bromide (EtBr), gentamycin (GEN), and chlorhexidine digluconate (CHX). We focused efforts on S. pseudintermedius as a pathogenic bacterium of concern within veterinary and human medicine. By combining data from checkerboard assays and EtBr efflux inhibition experiments, the hits 2-arylquinoline 1, dihydropyridine 6, and 2-phenyl-4-carboxy-quinoline 8 were considered the best EPIs for S. pseudintermedius. Overall, most of the compounds, except for 2-arylquinoline compound 2, were able to fully restore the susceptibility of S. pseudintermedius to CIP and synergize with GEN as well, while the synergistic effect with CHX was less significant and often did not show a dose-dependent effect. These are valuable data for medicinal chemistry optimization of EPIs for S. pseudintermedius and lay the foundation for further studies on successful EPIs to treat staphylococcal infections.
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Dengue virus (DENV), a mosquito-borne flavivirus, continues to be a major public health threat in many countries and no approved antiviral therapeutics are available yet. In this work, we designed and synthesized a series of sulfonyl anthranilic acid (SAA) derivatives using a ligand-based scaffold morphing approach of the 2,1-benzothiazine 2,2-dioxide core, previously used by us to develop DENV polymerase inhibitors resulting devoid of any cell-based antiviral activity. Several derivatives based on the new SAA chemotype exhibited potent inhibition against DENV infection in the cell-based assay but did not inhibit DENV NS5 polymerase activity in the in vitro de novo initiation and elongation assays. Notably, best compounds 26 and 39 showed EC50 values in the range of 0.54-1.36 µM against cells infected with the four dengue serotypes (DENV-1-4). Time-of-drug-addition assay revealed that analogue 26 is a post-entry replication inhibitor that appears to be specific for cells of primate origin, implicating a host target with a high barrier to resistance. In conclusion, SAA derivatives offer a valuable starting point for developing effective Dengue antiviral therapeutics.
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Vírus da Dengue , Dengue , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Dengue/tratamento farmacológico , Sorogrupo , Replicação ViralRESUMO
OBJECTIVES: To perform an in silico prediction of drug efflux pumps (EPs) in Staphylococcus pseudintermedius and investigate their role in conferring resistance to antibiotic and biocidal agents and biofilm formation. METHODS: A S. pseudintermedius efflux mutant was obtained by stimulating an isogenic line (ATCC 49444) with increasing concentrations of an efflux system substrate. Changes in antimicrobial susceptibility and biofilm-forming capability were evaluated in the presence/absence of the EP inhibitors (EPIs) thioridazine and reserpine and the efflux activity was assayed by fluorometry. Homologues of EPs of Staphylococcus aureus and Staphylococcus epidermidis were searched by exploratory GenBank investigations. Gene expression analyses and sequencing were then conducted on selected genes. RESULTS: Susceptibility to chlorhexidine, gentamicin and ciprofloxacin, but not enrofloxacin, was affected by the increased efflux and it was variably restored by the EPIs. The efflux mutant showed much greater biofilm formation that the original strain, which was significantly inhibited by thioridazine and reserpine at MIC/2. A high expression of norA, which was mgrA-independent, was found in the S. pseudintermedius efflux mutant, apparently regulated by an 11â bp deletion in its promoter region, whilst lmrB was transitorily overexpressed. icaA, which encodes the polysaccharide intercellular adhesin forming the extracellular matrix of staphylococcal biofilm, was also up-regulated. CONCLUSIONS: EPs, particularly NorA, are supposed to have complex involvement in multiple stages of resistance development. Overexpression of EPs appears to be correlated with a remarkable increase of S. pseudintermedius biofilm production; however, the regulatory mechanisms remain to be explored.
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Infecções Estafilocócicas , Tioridazina , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Reserpina/farmacologia , Infecções Estafilocócicas/veterináriaRESUMO
Antimicrobial resistance (AMR) represents a global health issue threatening our social lifestyle and the world economy. Efflux pumps are widely involved in AMR by playing a primary role in the development of specific mechanisms of resistance. In addition, they seem to be involved in the process of biofilm formation and maintenance, contributing to enhance the risk of creating superbugs difficult to treat. Accordingly, the identification of non-antibiotic molecules able to block efflux pumps, namely efflux pump inhibitors (EPIs), could be a promising strategy to counteract AMR and restore the antimicrobial activity of ineffective antibiotics. Herein, we enlarge the knowledge about the structure-activity relationship of 2-phenylquinoline Staphylococcus aureus NorA EPIs by reporting a new series of very potent C-6 functionalized derivatives. Best compounds significantly inhibited ethidium bromide efflux in a NorA-overexpressing S. aureus strain (SA-1199B) and strongly synergized at very low concentrations (0.20-0.78 µg/mL) with ciprofloxacin (CPX) against CPX-resistant S. aureus strains (SA-1199B and SA-K2378), as proved by checkerboard and time-kill experiments. In addition, some of these EPIs (9b and 10a) produced a post-antibiotic effect of 1.2 h and strongly enhanced antibiofilm activity of CPX against SA-1199B strain. Interestingly, at the concentrations used to reach synergy with CPX against resistant S. aureus strains, most of the EPI compounds did not show any human cell toxicity. Finally, by exploiting the recent released crystal structure of NorA, we observed that best EPI 9b highlighted a favourable docking pose, establishing some interesting interactions with key residues.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias , Biofilmes , Ciprofloxacina/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Plâncton/metabolismo , Staphylococcus aureusRESUMO
Pyridobenzothiazolone derivatives are a promising class of broad-spectrum antivirals. However, the mode of action of these compounds remains poorly understood. The HeE1-17Y derivative has already been shown to be a potent compound against a variety of flaviviruses of global relevance. In this work, the mode of action of HeE1-17Y has been studied for West Nile virus taking advantage of reporter replication particles (RRPs). Viral infectivity was drastically reduced by incubating the compound with the virus before infection, thus suggesting a direct interaction with the viral particles. Indeed, RRPs incubated with the inhibitor appeared to be severely compromised in electron microscopy analysis. HeE1-17Y is active against other enveloped viruses, including SARS-CoV-2, but not against two non-enveloped viruses, suggesting a virucidal mechanism that involves the alteration of the viral membrane.
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COVID-19 , Flavivirus , Vírus de RNA , Vírus , Antivirais/farmacologia , Humanos , SARS-CoV-2RESUMO
A selection of compounds from a proprietary library, based on chemical diversity and various biological activities, was evaluated as potential inhibitors of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a phenotypic-based screening assay. A compound based on a 2-phenylquinoline scaffold emerged as the most promising hit, with EC50 and CC50 values of 6 and 18 µM, respectively. The subsequent selection of additional analogues, along with the synthesis of ad hoc derivatives, led to compounds that maintained low µM activity as inhibitors of SARS-CoV-2 replication and lacked cytotoxicity at 100 µM. In addition, the most promising congeners also show pronounced antiviral activity against the human coronaviruses HCoV-229E and HCoV-OC43, with EC50 values ranging from 0.2 to 9.4 µM. The presence of a 6,7-dimethoxytetrahydroisoquinoline group at the C-4 position of the 2-phenylquinoline core gave compound 6g that showed potent activity against SARS-CoV-2 helicase (nsp13), a highly conserved enzyme, highlighting a potentiality against emerging HCoVs outbreaks.
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INTRODUCTION: Yellow fever virus (YFV) is a mosquito-borne flavivirus, endemic in 47 countries in Africa and South America, which causes febrile symptoms that can evolve in 15% of the patients to serious hemorrhagic conditions, liver injury, and multiorgan failure. Although a highly effective vaccine (YF-17D vaccine) is available, to date, no antiviral drugs have been approved for the prevention and treatment of YFV infections. AREAS COVERED: This review article focuses on the description of viral targets that have been considered within YFV and flavivirus drug discovery studies and on the most relevant candidates reported so far that elicit broad-spectrum inhibition against relevant strains and mutants of YFV. EXPERT OPINION: Considering the growing interest on (re)emerging vector-borne viral infections, it is expected that flavivirus drug discovery will quickly deliver potential candidates for clinical evaluation. Due to similarity among flaviviral targets, several candidates identified against different flaviviruses have shown broad-spectrum activity, thus exhibiting anti-YFV activity, as well. In this regard, it would be desirable to routinely include the assessment of antiviral activity against different YFV strains. On the other hand, the development of host targeting agents are still at an initial stage and deserve further focused efforts.
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Febre Amarela , Animais , Antivirais/farmacologia , Humanos , Febre Amarela/tratamento farmacológico , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/fisiologiaAssuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Antivirais/química , Inibidores Enzimáticos/química , Humanos , Vírus da Influenza A/enzimologia , Testes de Sensibilidade Microbiana , Subunidades Proteicas/efeitos dos fármacos , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Viruses are a continuing threat to global health. The lack or limited therapeutic armamentarium against some viral infections and increasing drug resistance issues make the search for new antiviral agents urgent. In recent years, a growing literature highlighted the use of triazolopyrimidine (TZP) heterocycles in the development of antiviral agents, with numerous compounds that showed potent antiviral activities against different RNA and DNA viruses. TZP core represents a privileged scaffold for achieving biologically active molecules, thanks to: i) the synthetic feasibility that allows to variously functionalize TZPs in the different positions of the nucleus, ii) the ability of TZP core to establish multiple interactions with the molecular target, and iii) its favorable pharmacokinetic properties. In the present review, after mentioning selected examples of TZP-based compounds with varied biological activities, we will focus on those antivirals that appeared in the literature in the last 10 years. Approaches used for their identification, the hit-to-lead studies, and the emerged structure-activity relationship will be described. A mention of the synthetic methodologies to prepare TZP nuclei will also be given. In addition, their mechanism of action, the binding mode within the biological target, and pharmacokinetic properties will be analyzed, highlighting the strengths and weaknesses of compounds based on the TZP scaffold, which is increasingly used in medicinal chemistry.
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Viroses , Vírus , Antivirais/química , Antivirais/farmacologia , Química Farmacêutica , Humanos , Relação Estrutura-AtividadeRESUMO
Antimicrobial resistance (AMR) is a complex threat to human health and, to date, it represents a hot topic in drug discovery. The use of non-antibiotic molecules to block resistance mechanisms is a powerful alternative to the identification of new antibiotics. Bacterial efflux pumps exert the early step of AMR development, allowing the bacteria to grow in presence of sub-inhibitory drug concentration and develop more specific resistance mechanisms. Thus, efflux pump inhibitors (EPIs) offer a great opportunity to fight AMR, potentially restoring antibiotic activity. Based on our experience in designing and synthesizing novel EPIs, herein, we retrieved information around quinoline and indole derivatives reported in literature on this topic. Thus, our aim was to collect all data around these promising classes of EPIs in order to delineate a comprehensive structure-activity relationship (SAR) around each core for different microbes. With this review article, we aim to help future research in the field in the discovery of new microbial EPIs with improved activity and a better safety profile.
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Antibacterianos , Bactérias , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias , Proteínas de Transporte , Quinolinas , Antibacterianos/química , Antibacterianos/uso terapêutico , Bactérias/química , Bactérias/metabolismo , Infecções Bacterianas/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Humanos , Quinolinas/química , Quinolinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
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.
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Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Quinazolinas/farmacologia , Quinolinas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Quinazolinas/síntese química , Quinazolinas/química , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR insights for this class of compounds and identify more potent derivatives, we designed and synthesized additional series of analogues to investigate the role of the substituents around the TZP core. To this aim, we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were functionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that showed both anti-PA-PB1 (IC50 = 19.5 µM) and anti-IAV activity (EC50 = 16 µM) at non-toxic concentrations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-CoV-2. The most promising compound 49 from this series displayed an EC50 value of 34.47 µM, highlighting the potential of the TPZ scaffold in the search for anti-CoV agents.
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Antivirais/farmacologia , Multimerização Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Chlorocebus aethiops , Cães , Desenho de Fármacos , Células HEK293 , Humanos , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Testes de Sensibilidade Microbiana , Pirimidinas/síntese química , SARS-CoV-2/efeitos dos fármacos , Triazóis/síntese química , Células VeroRESUMO
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. GâA 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.
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Rhodococcus equi , Rhodococcus , Antibacterianos/farmacologia , Etídio , Humanos , Testes de Sensibilidade Microbiana , Rhodococcus equi/genéticaRESUMO
Polymeric composites, where bioactive species are immobilized on inorganic nanostructured matrix, have received considerable attention as surfaces able to reduce bacterial adherence, colonization, and biofilm formation in implanted medical devices. In this work, potent in-house S. aureus NorA efflux pump inhibitors (EPIs), belonging to the 2-phenylquinoline class, were immobilized on nanometric alpha-zirconium phosphate (ZrP) taking into advantage of acid-base or intercalation reactions. The ZrP/EPI were used as filler of poly(lactide-co-glycolic acid) (PLGA) to obtain film composites with a homogeneous distribution of the ZrP/EPI fillers. As reference, PLGA films loaded with ZrP intercalated with thioridazine (TZ), that is recognized as both a NorA and biofilm inhibitor, and with the antibiotic ciprofloxacin (CPX) were prepared. Composite films were characterized by X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis. The ability of the composite films, containing ZrP/EPI, to inhibit biofilm formation was tested on Staphylococcus aureus ATCC 29213 and Staphylococcus epidermidis ATCC 12228, and it was compared with that of the composite loaded with ZrP/TZ. Finally, the antibacterial activity of CPX intercalated in ZrP was evaluated when used in combination with ZrP/EPI in the PLGA films.