RESUMO
Human and animal malaria parasites increase their host erythrocyte permeability to a broad range of solutes as mediated by parasite-associated ion channels. Molecular and pharmacological studies have implicated an essential role in parasite nutrient acquisition, but inhibitors suitable for development of antimalarial drugs are missing. Here, we generated a potent and specific drug lead using Plasmodium falciparum, a virulent human pathogen, and derivatives of MBX-2366, a nanomolar affinity pyridazinone inhibitor from a high-throughput screen. As this screening hit lacks the bioavailability and stability needed for in vivo efficacy, we synthesized 315 derivatives to optimize drug-like properties, establish target specificity, and retain potent activity against the parasite-induced permeability. Using a robust, iterative pipeline, we generated MBX-4055, a derivative active against divergent human parasite strains. MBX-4055 has improved oral absorption with acceptable in vivo tolerability and pharmacokinetics. It also has no activity against a battery of 35 human channels and receptors and is refractory to acquired resistance during extended in vitro selection. Single-molecule and single-cell patch-clamp indicate direct action on the plasmodial surface anion channel, a channel linked to parasite-encoded RhopH proteins. These studies identify pyridazinones as novel and tractable antimalarial scaffolds with a defined mechanism of action. SIGNIFICANCE STATEMENT: Because antimalarial drugs are prone to evolving resistance in the virulent human P. falciparum pathogen, new therapies are needed. This study has now developed a novel drug-like series of pyridazinones that target an unexploited parasite anion channel on the host cell surface, display excellent in vitro and in vivo ADME properties, are refractory to acquired resistance, and demonstrate a well defined mechanism of action.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Animais , Ânions/química , Ânions/metabolismo , Antimaláricos/farmacologia , Eritrócitos/metabolismo , Humanos , Nutrientes , Plasmodium falciparum/metabolismoRESUMO
The development of therapeutics for cytomegalovirus (CMV) infections, while progressing, has not matched the pace of new treatments of human immunodeficiency virus (HIV) infections; nevertheless, recent developments in the treatment of CMV infections have resulted in improved human health and perhaps will encourage the development of new therapeutic approaches. First, the deployment of ganciclovir and valganciclovir for both the prevention and treatment of CMV infections and disease in transplant recipients has been further improved with the licensure of the efficacious and less toxic letermovir. Regardless, late-onset CMV disease, specifically pneumonia, remains problematic. Second, the treatment of congenital CMV infections with valganciclovir has beneficially improved both hearing and neurologic outcomes, both fundamental advances for these children. In these pediatric studies, viral load was decreased but not eliminated. Thus, an important lesson learned from studies in both populations is the need for new antiviral agents and the necessity for combination therapies as has been shown to be beneficial in the treatment of HIV infections, among others. The development of monoclonal antibodies, sirtuins, and cyclopropovir may provide new treatment options.
Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Desenvolvimento de Medicamentos , Acetatos/administração & dosagem , Acetatos/efeitos adversos , Acetatos/uso terapêutico , Antivirais/farmacologia , Biomarcadores , Estudos Clínicos como Assunto , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/transmissão , Infecções por Citomegalovirus/virologia , Farmacorresistência Viral , Humanos , Transmissão Vertical de Doenças Infecciosas , Quinazolinas/administração & dosagem , Quinazolinas/efeitos adversos , Quinazolinas/uso terapêutico , Projetos de Pesquisa , Sirtuínas/administração & dosagem , Sirtuínas/efeitos adversos , Sirtuínas/uso terapêutico , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/métodos , Resultado do Tratamento , Carga ViralRESUMO
The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.IMPORTANCEP. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40 to 69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the PscF needle in T3SS secretion and translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. The results also support a model of direct interaction of the needle with PhA and suggest that, with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Sistemas de Secreção Tipo III/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação , Fenoxiacetatos/farmacologia , Pseudomonas aeruginosa/genética , Relação Estrutura-AtividadeRESUMO
Human adenovirus (HAdV) infection is common in the general population and can cause a range of clinical manifestations, among which pneumonia and keratoconjunctivitis are the most common. Although HAdV infections are mostly self-limiting, infections in immunocompromised individuals can be severe. No antiviral drug has been approved for treating adenoviruses. Filociclovir (FCV) is a nucleoside analogue which has successfully completed phase I human clinical safety studies and is now being developed for treatment of human cytomegalovirus (HCMV)-related disease in immunocompromised patients. In this report, we show that FCV is a potent broad-spectrum inhibitor of HAdV types 4 to 8, with 50% effective concentrations (EC50s) ranging between 1.24 and 3.6 µM and a 50% cytotoxic concentration (CC50) of 100 to 150 µM in human foreskin fibroblasts (HFFs). We also show that the prophylactic oral administration of FCV (10 mg/kg of body weight) 1 day prior to virus challenge and then daily for 14 days to immunosuppressed Syrian hamsters infected intravenously with HAdV6 was sufficient to prevent morbidity and mortality. FCV also mitigated tissue damage and inhibited virus replication in the liver. The 10-mg/kg dose had similar effects even when the treatment was started on day 4 after virus challenge. Furthermore, FCV administered at the same dose after intranasal challenge with HAdV6 partially mitigated body weight loss but significantly reduced pathology and virus replication in the lung. These findings suggest that FCV could potentially be developed as a pan-adenoviral inhibitor.
Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Infecções por Citomegalovirus , Infecções por Adenovirus Humanos/tratamento farmacológico , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Cricetinae , Infecções por Citomegalovirus/tratamento farmacológico , Humanos , Replicação ViralRESUMO
The most frequent ailment for which antibiotics are prescribed is otitis media (ear infections), which is most commonly caused by Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae Treatment of otitis media is complicated by the fact that the bacteria in the middle ear typically form biofilms, which can be recalcitrant to antibiotic treatment. Furthermore, bacterial respiratory infections can be greatly exacerbated by viral coinfection, which is particularly evidenced by the synergy between influenza and S. pneumoniae In this study, we sought to ascertain the in vivo efficacy of aminomethyl spectinomycin lead 1950, an effective antibacterial agent both in vitro and in vivo against Streptococcus pneumoniae in the context of complex respiratory infections and acute otitis media. A single dose of 1950 significantly reduced bacterial burden in the respiratory tract for all three pathogens, even when species were present in a coinfection model. Additionally, a single dose of 1950 effectively reduced pneumococcal acute otitis media from the middle ear. The agent 1950 also proved efficacious in the context of influenza-pneumococcal super infection. These data further support the development of this family of compounds as potential therapeutic agents against the common causes of complex upper respiratory tract infections and acute otitis media.
Assuntos
Infecções Respiratórias/tratamento farmacológico , Espectinomicina/uso terapêutico , Animais , Feminino , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Moraxella catarrhalis/efeitos dos fármacos , Moraxella catarrhalis/patogenicidade , Otite Média/tratamento farmacológico , Otite Média/microbiologia , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Espectinomicina/administração & dosagem , Espectinomicina/química , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/patogenicidadeRESUMO
To determine the mechanism of action of third-generation methylenecyclopropane nucleoside analogs (MCPNAs), DNA sequencing of herpes simplex virus 1 (HSV-1) isolates resistant to third-generation MCPNAs resulted in the discovery of G841S and N815S mutations in HSV-1 UL30. Purified HSV-1 UL30 or human cytomegalovirus (HCMV) UL54 was then subjected to increasing concentrations of MBX-2168-triphosphate (TP), with results demonstrating a 50% inhibitory concentration (IC50) of â¼200 µM, indicating that MBX-2168-TP does not inhibit the viral DNA polymerase. Further metabolic studies showed the removal of a moiety on the guanine ring of MBX-2168. Therefore, we hypothesized that enzymatic removal of a moiety at the 6-position of the guanine ring of third-generation MCPNAs is an essential step in activation. To test this hypothesis, pentostatin (deoxycoformycin [dCF]), an adenosine deaminase-like protein 1 (ADAL-1) inhibitor, was coincubated with MBX-2168. The results showed that dCF antagonized the effect of MBX-2168, with a >40-fold increase in the 50% effective concentration (EC50) at 50 µM dCF (EC50 of 63.1 ± 8.7 µM), compared with MBX-2168 alone (EC50 of 0.2 ± 0.1 µM). Purified ADAL-1 demonstrated time-dependent removal of the moiety on the guanine ring of MBX-2168-monophosphate (MP), with a Km of 17.5 ± 2.4 µM and a Vmax of 0.12 ± 0.04 nmol min-1 Finally, synguanol-TP demonstrated concentration-dependent inhibition of HSV-1 UL30 and HCMV UL54, with IC50s of 0.33 ± 0.16 and 0.38 ± 0.11 µM, respectively. We conclude that ADAL-1 is the enzyme responsible for removing the moiety from the guanine ring of MBX-2168-MP prior to conversion to a TP, the active compound that inhibits the viral DNA polymerase.
Assuntos
Adenosina Desaminase/metabolismo , Ciclopropanos/química , Ciclopropanos/farmacologia , Nucleosídeos/análogos & derivados , Nucleosídeos/farmacologia , Adenosina Desaminase/genética , Animais , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/patogenicidade , DNA Viral/genética , Guanina/análogos & derivados , Guanina/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/patogenicidade , Humanos , Análise de Sequência de DNA/métodos , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Filociclovir (MBX-400, cyclopropavir) is an antiviral agent with activity against cytomegalovirus (CMV). A phase 1, double-blind, randomized, placebo-controlled (3:1 ratio), single-center, multiple-ascending-dose trial was conducted to assess the safety, tolerability, and pharmacokinetics of filociclovir. Filociclovir (n = 18) or placebo (n = 6) was administered as a daily oral dose (100 mg, 350 mg, or 750 mg) for 7 days to normal healthy adults (ages, 25 to 65 years) who were monitored for 22 days. Safety assessments included clinical, laboratory, and electrocardiogram monitoring. Plasma and urine samplings were used to determine pharmacokinetic parameters. All study product-related adverse events were mild, most commonly gastrointestinal (17%), nervous system (11%), and skin and subcutaneous tissue (11%) disorders. One subject had reversible grade 3 elevation in serum creatinine and bilirubin, which was associated with an â¼1-log increase in plasma filociclovir exposure compared to levels for other subjects in the same (750-mg) cohort. No other serious adverse events were observed. Plasma exposures (area under the concentration-time curve from 0 to 24 h [AUC0-24]) on days 1 and 7 were similar, suggesting negligible dose accumulation. There was a sublinear increase in plasma exposure with dose, which plateaued at the daily dose of 350 mg. The amount of filociclovir recovered in the urine remained proportional to plasma exposure (AUC). Doses as low as 100 mg achieved plasma concentrations sufficient to inhibit CMV in vitro (This study has been registered at ClinicalTrials.gov under identifier NCT02454699.).
Assuntos
Antivirais/efeitos adversos , Antivirais/farmacocinética , Citomegalovirus/efeitos dos fármacos , Adulto , Idoso , Antivirais/sangue , Antivirais/uso terapêutico , Citomegalovirus/patogenicidade , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Meia-Vida , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Enantiomeric 3-deaza-1',6'-isoneplanocins (C-3 unsubstituted 7a/7b and C-3 with a bromine 8a/8b) lacking the 4'-hydroxymethyl as mechanistically designed anti-viral targets have been prepared by utilizing the Ullmann reaction. Anti-Ebola properties were found for the D-like 7a and 8a and L-like 8b. All four products showed effects against human cytomegalovirus while D-like 7a/8a affected measles; 7a was effective versus norovirus and 8a inhibited Pichinde. Both 7a and 8a produced SAHase inhibitory effects. However, the anti-EBOV activity of 7a and 8a cannot be readily correlated with this observation due with their contrasting IC50 values (8aâ¯>â¯7a). It is to be noted that 7b showed no effects on this enzyme and 8b was minimally inhibitory. These results offer preliminary insight into the differing mechanisms of action of D- and L- like structures and enlighten structural features to guide additional antiviral agent pursuit in the isoneplanocin series.
Assuntos
Adenosina/análogos & derivados , Antivirais/síntese química , Adenosina/síntese química , Adenosina/farmacologia , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Ebolavirus/efeitos dos fármacos , Eritrócitos/enzimologia , Humanos , Norovirus/efeitos dos fármacos , Coelhos , EstereoisomerismoRESUMO
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
Bacterial sexually transmitted infections are widespread and common, with Neisseria gonorrhoeae (gonorrhea) and Chlamydia trachomatis (chlamydia) being the two most frequent causes. If left untreated, both infections can cause pelvic inflammatory disease, infertility, ectopic pregnancy, and other sequelae. The recommended treatment for gonorrhea is ceftriaxone plus azithromycin (to empirically treat chlamydial coinfections). Antibiotic resistance to all existing therapies has developed in gonorrheal infections. The need for new antibiotics is great, but the pipeline for new drugs is alarmingly small. The aminomethyl spectinomycins, a new class of semisynthetic analogs of the antibiotic spectinomycin, were developed on the basis of a computational analysis of the spectinomycin binding site of the bacterial 30S ribosome and structure-guided synthesis. The compounds display particular potency against common respiratory tract pathogens as well as the sexually transmitted pathogens that cause gonorrhea and chlamydia. Here, we demonstrate the in vitro potencies of several compounds of this class against both bacterial species; the compounds displayed increased potencies against N. gonorrhoeae compared to that of spectinomycin and, significantly, demonstrated activity against C. trachomatis that is not observed with spectinomycin. Efficacies of the compounds were compared to those of spectinomycin and gentamicin in a murine model of infection caused by ceftriaxone/azithromycin-resistant N. gonorrhoeae; the aminomethyl spectinomycins significantly reduced the colonization load and were as potent as the comparator compounds. In summary, data produced by this study support aminomethyl spectinomycins as a promising replacement for spectinomycin and antibiotics such as ceftriaxone for treating drug-resistant gonorrhea, with the added benefit of treating chlamydial coinfections.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Chlamydia/tratamento farmacológico , Chlamydia trachomatis/efeitos dos fármacos , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/efeitos dos fármacos , Doenças Bacterianas Sexualmente Transmissíveis/tratamento farmacológico , Espectinomicina/análogos & derivados , Espectinomicina/uso terapêutico , Animais , Azitromicina/farmacologia , Ceftriaxona/farmacologia , Infecções por Chlamydia/microbiologia , Coinfecção/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla , Feminino , Gentamicinas/uso terapêutico , Gonorreia/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Subunidades Ribossômicas Menores de Bactérias/efeitos dos fármacosRESUMO
Synthetically combining the C-4' side-chain structural features of the antiviral candidates 5'-methylaristeromycin and 5'-homoaristeromycin into a diastereomeric pair of C-4' side-chain dihydroxylated aristeromycins (6 and 7) is reported. Broad antiviral analyses of the both targets found promising effects towards HBV (6, 6.7⯵M and 7, 7.74⯵M) and HCMV (only 7, 0.72⯵M). No other activity was found. Neither of the diastereomers was cytotoxic in the assays performed.
Assuntos
Adenosina/análogos & derivados , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Adenosina/síntese química , Adenosina/química , Adenosina/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
A convenient stereospecific synthesis of 6'-fluoro-3-deazaneplanocin (6) has been accomplished from d-ribose in 15 steps. It is reported to possess significant activity towards Ebola (Zaire, Vero, µM: EC50â¯<â¯0.36; CC50 125; SIâ¯>â¯347) with moderate inhibition of the target enzyme (S-adenosylhomocysteine hydrolase), which did not correlate directly with its anti-Ebola effects. Compound 6, with limited cytotoxicity, also displayed activity against measles, H1N1 and Pichinde.
Assuntos
Adenosina/análogos & derivados , Antivirais/síntese química , Adenosina/síntese química , Adenosina/química , Adenosina/farmacologia , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Chlorocebus aethiops , Ebolavirus/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Concentração Inibidora 50 , Células VeroRESUMO
Pseudomonas aeruginosa is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or microabscess formation. The rising incidence of multidrug resistance among P. aeruginosa isolates has increased both the economic burden and the morbidity and mortality associated with P. aeruginosa disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells in vitro In this study, we used a mouse model of P. aeruginosa abscess formation to test the in vivo efficacy of these compounds against the P. aeruginosa type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of the abscesses, as neutrophil depletion resulted in an increased abscess size, the formation of dermonecrotic lesions on the skin, and the dissemination of P. aeruginosa to internal organs. Consistent with the specificity of the T3SS-neutrophil interaction, P. aeruginosa bacteria lacking a functional T3SS were fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in the immune clearance of the bacteria. Finally, a P. aeruginosa strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together, our results further define the role of type III secretion in murine abscess formation and demonstrate the in vivo efficacy of phenoxyacetamide inhibitors in P. aeruginosa infection.
Assuntos
Abscesso/microbiologia , Antibacterianos/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Abscesso/tratamento farmacológico , Abscesso/patologia , Animais , Antibacterianos/química , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Camundongos Endogâmicos C57BL , Neutropenia/microbiologia , Neutrófilos/patologia , Fenoxiacetatos/química , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Sistemas de Secreção Tipo III , Fatores de Virulência/metabolismoRESUMO
Objectives: New drug regimens employing combinations of existing and experimental antimicrobial agents are needed to shorten treatment of tuberculosis (TB) in humans. The spectinamides are narrow-spectrum semisynthetic analogues of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis . Spectinamides, including lead 1599, have been previously shown to exhibit a promising therapeutic profile in mice as single agents. Here we explore the in vivo activity of lead spectinamides when combined with other agents. Methods: The efficacy of 1599 or 1810 was tested in combination in three increasingly advanced TB mouse models. Mice were infected by aerosol and allowed to establish acute or chronic infection, followed by treatment (≤4â weeks) with the spectinamides alone or in two- and three-drug combination regimens with existing and novel therapeutic agents. Bacteria were enumerated from lungs by plating for cfu. Results: Herein we show the following: (i) 1599 exhibits additive or synergistic activity with most of the first-line agents; (ii) 1599 in combination with rifampicin and pyrazinamide or with bedaquiline and pyrazinamide promotes significantly improved efficacy in the high-dose aerosol model; (iii) 1599 enhances efficacy of rifampicin or pyrazinamide in chronically infected BALB/c mice; and (iv) 1599 is synergistic when administered in combination with rifampicin and pyrazinamide in the C3HeB/FeJ mouse model showing caseous necrotic pulmonary lesions. Conclusions: Spectinamides were effective partner agents for multiple anti-TB agents including bedaquiline, rifampicin and pyrazinamide. None of these in vivo synergistic interactions was predicted from in vitro MIC chequerboard assays. These data support further development of the spectinamides as combination partners with existing and experimental anti-TB agents.
Assuntos
Antituberculosos/uso terapêutico , Espectinomicina/química , Espectinomicina/uso terapêutico , Tuberculose/tratamento farmacológico , Animais , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/uso terapêutico , Quinolinas/uso terapêutico , Rifampina/uso terapêutico , Tuberculose/microbiologiaRESUMO
Cyclopropavir (CPV) is a promising antiviral drug against human cytomegalovirus (HCMV). As with ganciclovir (GCV), the current standard for HCMV treatment, activation of CPV requires multiple steps of phosphorylation and is enantioselective. We hypothesized that the resulting CPV triphosphate (CPV-TP) would stereoselectively target HCMV DNA polymerase and terminate DNA synthesis. To test this hypothesis, we synthesized both enantiomers of CPV-TP [(+) and (-)] and investigated their action on HCMV polymerase. Both enantiomers inhibited HCMV polymerase competitively with dGTP, with (+)-CPV-TP exhibiting a more than 20-fold lower apparent Ki than (-)-CPV-TP. Moreover, (+)-CPV-TP was a more potent inhibitor than GCV-TP. (+)-CPV-TP also exhibited substantially lower apparent Km and somewhat higher apparent kcat values than (-)-CPV-TP and GCV-TP for incorporation into DNA by the viral polymerase. As is the case for GCV-TP, both CPV-TP enantiomers behaved as nonobligate chain terminators, with the polymerase terminating DNA synthesis after incorporation of one additional nucleotide. These results elucidate how CPV-TP acts on HCMV DNA polymerase and help explain why CPV is more potent against HCMV replication than GCV.
Assuntos
Antivirais/farmacologia , Ciclopropanos/farmacologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Replicação do DNA/efeitos dos fármacos , DNA Viral/genética , Farmacorresistência Viral/genética , Guanina/farmacologia , Cinética , Estrutura Molecular , Fosforilação/efeitos dos fármacosRESUMO
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
The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas da Matriz Viral/química , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Cinética , Proteínas Mutantes , Neuraminidase/química , Neuraminidase/metabolismo , Ligação Proteica , Proteínas da Matriz Viral/metabolismoRESUMO
BACKGROUND: The current Ebola virus (EBOV) outbreak has highlighted the troubling absence of available antivirals or vaccines to treat infected patients and stop the spread of EBOV. The EBOV glycoprotein (GP) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-EBOV drugs. We report the identification of 2 novel EBOV inhibitors targeting viral entry. METHODS: To identify small molecule inhibitors of EBOV entry, we carried out a cell-based high-throughput screening using human immunodeficiency virus-based pseudotyped viruses expressing EBOV-GP. Two compounds were identified, and mechanism-of-action studies were performed using immunoflourescence, AlphaLISA, and enzymatic assays for cathepsin B inhibition. RESULTS: We report the identification of 2 novel entry inhibitors. These inhibitors (1) inhibit EBOV infection (50% inhibitory concentration, approximately 0.28 and approximately 10 µmol/L) at a late stage of entry, (2) induce Niemann-Pick C phenotype, and (3) inhibit GP-Niemann-Pick C1 (NPC1) protein interaction. CONCLUSIONS: We have identified 2 novel EBOV inhibitors, MBX2254 and MBX2270, that can serve as starting points for the development of an anti-EBOV therapeutic agent. Our findings also highlight the importance of NPC1-GP interaction in EBOV entry and the attractiveness of NPC1 as an antifiloviral therapeutic target.
Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Chlorocebus aethiops , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Proteína C1 de Niemann-Pick , Ligação Proteica/efeitos dos fármacos , Células Vero , Internalização do Vírus/efeitos dos fármacosRESUMO
Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Cristalografia por Raios X , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Humanos , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Internalização do Vírus/efeitos dos fármacosRESUMO
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.