RESUMO
Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.
Assuntos
Antifúngicos , Aspergillus fumigatus , Farmacorresistência Fúngica , Ácidos Graxos Insaturados , Testes de Sensibilidade Microbiana , Triazóis , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Antifúngicos/farmacologia , Triazóis/farmacologia , Camundongos , Animais , Ácidos Graxos Insaturados/farmacologia , Humanos , Ensaios de Triagem em Larga Escala , Sinergismo Farmacológico , Rhizopus/efeitos dos fármacos , Rhizopus/genética , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Piperidinas/farmacologia , Modelos Animais de Doenças , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Aspergilose Pulmonar Invasiva/microbiologiaRESUMO
Multi-drug-resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and disseminate in the host. The Type 3 secretion system (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, composed of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this article, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly. The assay demonstrates a favorable signal/noise ratio (13.6) and robustness (Z' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Humanos , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/genética , Sistemas de Secreção Tipo III , MamíferosRESUMO
BACKGROUND: Efflux pump inhibitors (EPIs) offer an attractive therapeutic option when combined with existing classes. However, their optimal dosing strategies are unknown. METHODS: MICs of ciprofloxacin (CIP)+/-chlorpromazine, phenylalanine-arginine ß naphthylamide (PAßN) and a developmental molecule MBX-4191 were determined and the pharmacodynamics (PD) was studied in an in vitro model employing Escherichia coli MG1655 and its isogenic MarR mutant (I1147). Exposure ranging experiments were performed initially then fractionation. Changes in bacterial load and population profiles were assessed. Strains recovered after EPI simulations were studied by WGS. RESULTS: The CIPMICs for E. coli MG1655 and I1147 were 0.08 and 0.03 mg/L. Chlorpromazine at a concentration of 60 mg/L, PAßN concentrations of 30 mg/L and MBX-4191 concentrations of 0.5-1.0 mg/L reduced CIP MICs for I1147 and enhanced bacterial killing. Using CIP at an AUC of 1.2 mg·h/L, chlorpromazine AUC was best related to reduction in bacterial load at 24 h, however, when the time drug concentration was greater than 25 mg/L (Tâ>â25 mg/L) chlorpromazine was also strongly related to the effect. For PaßN with CIP AUC, 0.6 mg·h/L PaßN AUC was best related to a reduction in bacterial load. MBX-4191Tâ>â0.5-0.75 mg·h/L was best related to reduction in bacterial load. Changes in population profiles were not seen in experiments of ciprofloxacinâ+âEPIs. WGS of recovered strains from simulations with all three EPIs showed mutations in gyrA, gyrB or marR. CONCLUSIONS: AUC was the pharmacodynamic driver for chlorpromazine and PAßN while Tâ>âthreshold was the driver for MBX-4191 and important in the activity of chlorpromazine and PAßN. Changes in population profiles did not occur with combinations of ciprofloxacinâ+âEPIs, however, mutations in gyrA, gyrB and marR were detected.
Assuntos
Clorpromazina , Escherichia coli , Escherichia coli/genética , Clorpromazina/farmacologia , Farmacorresistência Bacteriana Múltipla , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Testes de Sensibilidade Microbiana , Farmacorresistência BacterianaRESUMO
Only three classes of contemporary antifungal drugs are routinely utilized in the clinic against filamentous fungal pathogens such as Aspergillus fumigatus. High-throughput phenotypic screens to identify small molecules with activity against filamentous fungi remain challenging due to the hyphal, biofilm-like growth morphology of these important organisms. In this chapter, we describe a protocol for utilizing a bioluminescent A. fumigatus strain for identifying small molecules that potentiate the activity of the triazole antifungal drug fluconazole. The assay holds great promise for identifying small molecules with activity against filamentous fungal pathogens.
Assuntos
Antifúngicos , Aspergillus fumigatus , Antifúngicos/farmacologia , Fungos , Triazóis , Hifas , Testes de Sensibilidade MicrobianaRESUMO
Human cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index >30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Humanos , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/etiologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Ganciclovir/farmacologia , Hospedeiro ImunocomprometidoRESUMO
The increasing prevalence of multidrug-resistant (MDR) bacterial infections has created a crucial need for new therapeutics that avoid or minimize existing resistance mechanisms. In this review, we describe the development of novel classes of small-molecule adjunctive agents targeting either a bacterial virulence factor, the Pseudomonas aeruginosa type III secretion system (T3SS), or an intrinsic resistance factor, resistance-nodulation-cell division superfamily (RND) efflux pumps of the Enterobacteriaceae. These agents are designed to be administered with antibacterials to improve their efficacy. T3SS inhibition rescues host innate immune system cells from injection with bacterial toxins, whereas RND efflux pump inhibition increases antibiotic susceptibility, in both cases improving the efficacy of the combined antibacterial.
Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Proteínas de Membrana Transportadoras/metabolismo , Sistemas de Secreção Tipo III/antagonistas & inibidores , Animais , Proteínas de Bactérias/metabolismo , Humanos , Sistemas de Secreção Tipo III/metabolismoRESUMO
Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.
Assuntos
Neisseria gonorrhoeae/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Células CACO-2 , Feminino , Gonorreia/microbiologia , Gonorreia/prevenção & controle , Humanos , Camundongos , Neisseria gonorrhoeae/genética , Biossíntese de Proteínas/genética , Inibidores da Síntese de Proteínas/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
BACKGROUND: The bacterial cell envelope is comprised of the cell membrane and the cell wall. The bacterial cell wall provides rigidity to the cell and protects the organism from potential harmful substances also. Cell wall biosynthesis is an important physiological process for bacterial survival and thus has been a primary target for the development of antibacterials. Antimicrobial peptides that target bacterial cell wall assembly are abundant and many bind to the essential cell wall precursor molecule Lipid II. METHODS: We describe the structure-to-activity (SAR) relationship of an antimicrobial peptide-derived small molecule 7771-0701 that acts as a novel agent against cell wall biosynthesis. Derivatives of compound 7771-0701 (2-[(1E)-3-[(2E)-5,6-dimethyl-3-(prop-2-en-1-yl)-1,3-benzothiazol-2-ylidene]prop-1-en-1-yl]-1,3,3-trimethylindol-1-ium) were generated by medicinal chemistry guided by Computer-Aided Drug Design and NMR. Derivatives were tested for antibacterial activity and Lipid II binding. RESULTS: Our results show that the N-alkyl moiety is subject to change without affecting functionality and further show the functional importance of the sulfur in the scaffold. The greatest potency against Gram-positive bacteria and Lipid II affinity was achieved by incorporation of a bromide at the R3 position of the benzothiazole ring. CONCLUSION: We identify optimized small molecule benzothiazole indolene scaffolds that bind to Lipid II for further development as antibacterial therapeutics.
Assuntos
Antibacterianos/farmacologia , Benzotiazóis/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Antibacterianos/síntese química , Antibacterianos/química , Benzotiazóis/síntese química , Benzotiazóis/química , Parede Celular/efeitos dos fármacos , Desenho de Fármacos , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
Lipid II is an essential precursor of bacterial cell wall biosynthesis and an attractive target for antibiotics. Lipid II is comprised of specialized lipid (bactoprenol) linked to a hydrophilic head group consisting of a peptidoglycan subunit (N-acetylglucosamine (GlcNAc)-N-acetylmuramic acid (MurNAc) disaccharide coupled to a short pentapeptide moiety) via a pyrophosphate. We previously identified a (E)-2,4-bis(4-bromophenyl)-6-(4-(dimethylamino)styryl)pyrylium boron tetrafluoride salt, termed 6jc48-1, that interacts with the MurNAc moiety, the phosphate cage and the isoprenyl tail of Lipid II. Here, we report on the structure-activity relationship of 6jc48-1 derivatives obtained by de novo chemical synthesis. Our results indicate that bacterial killing is positively driven by bi-phenyl stacking with peptidoglycan units. Replacement of bromides by fluorides resulted in activity against S. aureus without affecting Lipid II binding and cytotoxicity. Antibacterial activity was affected negatively by extended interaction of the scaffold with Lipid II isoprenyl units.
Assuntos
Desenvolvimento de Medicamentos/métodos , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Relação Estrutura-Atividade , Uridina Difosfato Ácido N-Acetilmurâmico/químicaRESUMO
We identified low-molecular weight compounds derived from the antimicrobial peptide human neutrophil peptide-1 that bind to Lipid II, an essential precursor of bacterial cell wall biosynthesis. These compounds act as antibacterials on multiple biosynthesis pathways with specificity against gram-positive organisms. Here, we have tested a small subset of our most promising leads against the bacterium Clostridium perfringens and sporozoites of Eimeria tenella, an intracellular protozoan parasite that causes intestinal disease in poultry. We found one compound, 1611-0203 (2-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(2-hydroxyphenoxy)phenyl]phenoxy}phenol), specifically to inhibit growth of both agents out of all compounds tested. Additionally, compound 1611-0203 inhibits Staphylococcus aureus and Enterococcus spp. Mechanism-of-action studies further reveal that 1611-0203 affects cell wall biosynthesis and inhibits additional biosynthetic pathways. Combined, our results indicate that compounds such as 1611-0203 have therapeutic potential to act as anti-infectives against various organisms simultaneously.
Efectos anti infecciosos pleiotrópicos de los compuestos antimicrobianos derivados de la defensina. Se identificaron compuestos de bajo peso molecular derivados del péptido antimicrobiano, péptido neutrófilico humano 1 que se unen al lípido II, que es un precursor esencial de la biosíntesis de la pared celular bacteriana. Estos compuestos actúan como antibacterianos en múltiples vías de biosíntesis con especificidad contra organismos Gram positivos. En este estudio se probó un pequeño subconjunto de los proyectos más prometedores contra la bacteria Clostridium perfringens y los esporozoitos de Eimeria tenella, que es un parásito protozoario intracelular que causa enfermedad intestinal en aves comerciales. Se encontró un compuesto, 1611-0203 (2-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(2-hidroxifenoxi)fenil]fenoxi}fenol), específicamente para inhibir el crecimiento de ambos agentes de todos los compuestos probados. Además, el compuesto 1611-0203 inhibe a Staphylococcus aureus y Enterococcus spp. Los estudios del mecanismo de acción revelan además que 1611-0203 afecta la biosíntesis de la pared celular e inhibe las vías biosintéticas adicionales. Estos resultados combinados indican que los compuestos como el 1611-0203 tienen potencial terapéutico para actuar como anti infecciosos contra varios organismos simultáneamente.
Assuntos
Anti-Infecciosos/farmacologia , Clostridium perfringens/efeitos dos fármacos , Defensinas/farmacologia , Eimeria tenella/efeitos dos fármacos , Enterococcus/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular , Galinhas , Fibroblastos/efeitos dos fármacosRESUMO
Resistance-nodulation-division (RND) superfamily efflux pumps play a major role in multidrug resistance (MDR) of Gram-negative pathogens by extruding diverse classes of antibiotics from the cell. There has been considerable interest in developing efflux pump inhibitors (EPIs) of RND pumps as adjunctive therapies. The primary challenge in EPI discovery has been the highly hydrophobic, poly-specific substrate binding site of the target. Recent findings have identified the hydrophobic trap, a narrow phenylalanine-lined groove in the substrate-binding site, as the "Achilles heel" of the RND efflux pumps. In this review, we will examine the hydrophobic trap as an EPI target and two chemically distinct series of EPIs that bind there.
Assuntos
Proteínas de Bactérias/química , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Família MultigênicaRESUMO
AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.IMPORTANCE Antibiotic resistance is a major public health concern. In Gram-negative bacteria, overexpression of the AcrAB-TolC multidrug efflux system confers resistance to clinically useful drugs. Here, we show that loss of AcrB efflux function causes loss of virulence in Salmonella enterica serovar Typhimurium. This is due to the reduction of bacterial factors necessary for infection, which is likely to be caused by the retention of noxious molecules inside the bacterium. We also show that, in contrast to loss of AcrB protein, loss of efflux does not induce overexpression of other efflux pumps from the same family. This indicates that there are differences between loss of efflux protein and loss of efflux that make gene deletion mutants unsuitable for studying the biological function of membrane transporters. Understanding the biological role of AcrB will help to assess the risks of targeting efflux pumps as a strategy to combat antibiotic resistance.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Salmonella typhimurium/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Benzimidazóis/metabolismo , Transporte Biológico , Modelos Animais de Doenças , Endocitose , Células Epiteliais/microbiologia , Etídio/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Ilhas Genômicas , Lepidópteros , Proteínas de Membrana Transportadoras/genética , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Salmonelose Animal , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Virulência , Fatores de Virulência/genéticaRESUMO
Recently we described a novel di-benzene-pyrylium-indolene (BAS00127538) inhibitor of Lipid II. BAS00127538 (1-Methyl-2,4-diphenyl-6-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)pyryl-1-ium) tetrafluoroborate is the first small molecule Lipid II inhibitor and is structurally distinct from natural agents that bind Lipid II, such as vancomycin. Here, we describe the synthesis and biological evaluation of 50 new analogs of BAS00127538 designed to explore the structure-activity relationships of the scaffold. The results of this study indicate an activity map of the scaffold, identifying regions that are critical to cytotoxicity, Lipid II binding and range of anti-bacterial action. One compound, 6jc48-1, showed significantly enhanced drug-like properties compared to BAS00127538. 6jc48-1 has reduced cytotoxicity, while retaining specific Lipid II binding and activity against Enterococcus spp. in vitro and in vivo. Further, this compound showed a markedly improved pharmacokinetic profile with a half-life of over 13 hours upon intravenous and oral administration and was stable in plasma. These results suggest that scaffolds like that of 6jc48-1 can be developed into small molecule antibiotic drugs that target Lipid II.
Assuntos
Antibacterianos/farmacologia , Lipídeos/antagonistas & inibidores , Animais , Proteínas Sanguíneas/metabolismo , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/efeitos dos fármacos , Simulação de Dinâmica Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
We previously reported the synthesis and biological activity of a series of cationic bis-indoles with potent, broad-spectrum antibacterial properties. Here, we describe mechanism of action studies to test the hypothesis that these compounds bind to DNA and that this target plays an important role in their antibacterial outcome. The results reported here indicate that the bis-indoles bind selectively to DNA at A/T-rich sites, which is correlated with the inhibition of DNA and RNA synthesis in representative Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) organisms. Further, exposure of E. coli and S. aureus to representative bis-indoles resulted in induction of the DNA damage-inducible SOS response. In addition, the bis-indoles were found to be potent inhibitors of cell wall biosynthesis; however, they do not induce the cell wall stress stimulon in S. aureus, suggesting that this pathway is inhibited by an indirect mechanism. In light of these findings, the most likely basis for the observed activities of these compounds is their ability to bind to the minor groove of DNA, resulting in the inhibition of DNA and RNA synthesis and other secondary effects.
Assuntos
Antibacterianos/farmacologia , DNA/metabolismo , Indóis/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Escherichia coli/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Indóis/química , Indóis/metabolismo , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Terapia de Alvo Molecular , Resposta SOS em Genética/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
Cytomegalovirus (CMV) is a ubiquitous human pathogen that increases the morbidity and mortality of immunocompromised individuals. The current FDA-approved treatments for CMV infection are intended to be virus specific, yet they have significant adverse side effects, including nephrotoxicity and hematological toxicity. Thus, there is a medical need for safer and more effective CMV therapeutics. Using a high-content screen, we identified the cardiac glycoside convallatoxin as an effective compound that inhibits CMV infection. Using a panel of cardiac glycoside variants, we assessed the structural elements critical for anti-CMV activity by both experimental and in silico methods. Analysis of the antiviral effects, toxicities, and pharmacodynamics of different variants of cardiac glycosides identified the mechanism of inhibition as reduction of methionine import, leading to decreased immediate-early gene translation without significant toxicity. Also, convallatoxin was found to dramatically reduce the proliferation of clinical CMV strains, implying that its mechanism of action is an effective strategy to block CMV dissemination. Our study has uncovered the mechanism and structural elements of convallatoxin, which are important for effectively inhibiting CMV infection by targeting the expression of immediate-early genes. IMPORTANCE: Cytomegalovirus is a highly prevalent virus capable of causing severe disease in certain populations. The current FDA-approved therapeutics all target the same stage of the viral life cycle and induce toxicity and viral resistance. We identified convallatoxin, a novel cell-targeting antiviral that inhibits CMV infection by decreasing the synthesis of viral proteins. At doses low enough for cells to tolerate, convallatoxin was able to inhibit primary isolates of CMV, including those resistant to the anti-CMV drug ganciclovir. In addition to identifying convallatoxin as a novel antiviral, limiting mRNA translation has a dramatic impact on CMV infection and proliferation.
Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus/efeitos dos fármacos , Metionina/metabolismo , Estrofantinas/farmacologia , Antivirais/química , Transporte Biológico Ativo/efeitos dos fármacos , Glicosídeos Cardíacos/química , Glicosídeos Cardíacos/farmacologia , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Genes Precoces/efeitos dos fármacos , Genes Virais/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Estrofantinas/química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
The rise of multidrug resistant (MDR) Gram-negative pathogens complicates our ability to treat bacterial infections with antibiotics. MDR efflux pumps play a major role in the acquisition and expression of the MDR phenotype. The major MDR efflux pumps in Gram-negative pathogens are the resistance-nodulation-division (RND) superfamily pumps. Efflux pump inhibitors (EPIs) that target RND superfamily pumps could play an important role in the clinic as an adjunctive therapy to increase antibiotic efficacy, decrease resistance, and attenuate virulence in Gram-negative pathogens. Here, we review recent advances in the discovery and structurally enabled optimization of a novel series of RND-targeting pyranopyridine EPIs currently in the early stages of lead optimization.
Assuntos
Antibacterianos/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Piranos/farmacologia , Piridinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Relação Estrutura-AtividadeRESUMO
The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.
Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Piridinas/farmacologia , Antibacterianos/química , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Estrutura Terciária de Proteína , Piranos/química , Piranos/farmacologia , Piridinas/químicaRESUMO
Herein, we describe the antifungal evaluation of 43 bisamidine compounds, of which 26 are new, having the scaffold [Am]-[HetAr]-[linker]-[HetAr]-[Am], in which [Am] is a cyclic or acyclic amidine group, [linker] is a benzene, pyridine, pyrimidine, pyrazine ring, or an aliphatic chain of two to four carbon, and [HetAr] is a 5,6-bicyclic heterocycle such as indole, benzimidazole, imidazopyridine, benzofuran, or benzothiophene. In the head-to-head series the two [HetAr] units are oriented such that the 5-membered rings are connected through the linker, and in the head-to-tail series, one of the [HetAr] systems is connected through the 6-membered ring; additionally, in some of the head-to-tail compounds, the [linker] is omitted. Many of these compounds exhibited significant antifungal activity against Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans (MIC ⩽ 4 µg/ml). The most potent compounds, for example, P10, P19 and P34, are comparable in antifungal activities to amphotericin B (MIC 0.125 µg/ml). They exhibited rapid fungicidal activity (>3 log10 decrease in cfu/ml in 4h) at concentrations equivalent to 4× the MIC in time kill experiments. The bisamidines strongly inhibited DNA, RNA and cell wall biosynthesis in C. albicans in macromolecular synthesis assays. However, the half-maximal inhibitory concentration for DNA synthesis was approximately 30-fold lower than those for RNA and cell wall biosynthesis. Fluorescence microscopy of intact cells of C. albicans treated with a bisamidine exhibited enhanced fluorescence in the presence of DNA, demonstrating that the bisamidine was localized to the nucleus. The results of this study show that bisamidines are potent antifungal agents with rapid fungicidal activity, which is likely to be the result of their DNA-binding activity. Although it was difficult to obtain a broad-spectrum antifungal compound with low cytotoxicity, some of the compounds (e.g., P9, P14 and P43) exhibited favorable CC50 values against HeLa cells and maintained considerable antifungal activity.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Furanos/química , Furanos/síntese química , HumanosRESUMO
Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 µM). The most potent SABA analogs had MICs of 0.5 to 7.0 µM (0.2 to 3.0 µg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII.
Assuntos
Antibacterianos/farmacologia , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Acetilcoenzima A/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologiaRESUMO
Multidrug resistance (MDR) in Gram-negative pathogens, such as the Enterobacteriaceae and Pseudomonas aeruginosa, poses a significant threat to our ability to effectively treat infections caused by these organisms. A major component in the development of the MDR phenotype in Gram-negative bacteria is overexpression of Resistance-Nodulation-Division (RND)-type efflux pumps, which actively pump antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of RND-type efflux pump have been reported in the literature, and at least three of these EPI series were optimized in a pre-clinical development program. However, none of these compounds have been tested in the clinic. One of the major hurdles to the development of EPIs has been the lack of biochemical, computational, and structural methods that could be used to guide rational drug design. Here, we review recent reports that have advanced our understanding of the mechanism of action of several potent EPIs against RND-type pumps.