RESUMO
Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infections in infants and the immunocompromised, yet no efficient therapeutic exists. We have identified the AVG class of allosteric inhibitors of RSV RNA synthesis. Here, we demonstrate through biolayer interferometry and in vitro RNA-dependent RNA polymerase (RdRP) assays that AVG compounds bind to the viral polymerase, stalling the polymerase in initiation conformation. Resistance profiling revealed a unique escape pattern, suggesting a discrete docking pose. Affinity mapping using photoreactive AVG analogs identified the interface of polymerase core, capping, and connector domains as a molecular target site. A first-generation lead showed nanomolar potency against RSV in human airway epithelium organoids but lacked in vivo efficacy. Docking pose-informed synthetic optimization generated orally efficacious AVG-388, which showed potent efficacy in the RSV mouse model when administered therapeutically. This study maps a druggable target in the RSV RdRP and establishes clinical potential of the AVG chemotype against RSV disease.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Animais , Humanos , Camundongos , Conformação Molecular , RNA Polimerase Dependente de RNA , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/genéticaRESUMO
Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an in vitro RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hit-to-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of >1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with in silico pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratory-adapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.
Assuntos
Antivirais/farmacologia , RNA Polimerase Dependente de RNA/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Regulação Alostérica , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , RNA Polimerase Dependente de RNA/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Proteínas Virais/metabolismoRESUMO
We investigated the susceptibility of 10 enterovirus D68 (EV-D68) isolates (belonging to clusters A, B, and C) to (entero)virus inhibitors with different mechanisms of action. The 3C-protease inhibitors proved to be more efficient than enviroxime and pleconaril, which in turn were more effective than vapendavir and pirodavir. Favipiravir proved to be a weak inhibitor. Resistance to pleconaril maps to V69A in the VP1 protein, and resistance to rupintrivir maps to V104I in the 3C protease. A structural explanation of why both substitutions may cause resistance is provided.
Assuntos
Antivirais/farmacologia , Enterovirus Humano D/efeitos dos fármacos , Infecções por Enterovirus/virologia , Farmacorresistência Viral , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Oxidiazóis/farmacologia , Oxazóis , Receptores de Droga/química , Receptores de Droga/efeitos dos fármacos , Infecções Respiratórias/virologia , Proteínas Virais/química , Replicação Viral/efeitos dos fármacosRESUMO
We herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogs with antiviral or anticancer activity. Twenty-five symmetrical phosphorodiamidates were synthesized, bearing esterified l-Alanine (and in one case d-Alanine) in the prodrug moiety, each as single stereoisomer. The presence of an achiral phosphorus represents a potential advantage over the phosphoramidate ProTide approach, where diastereoisomeric mixtures are routinely obtained, and different biological profiles may be expected from the diastereoisomers. Optimization of the synthetic pathway allowed us to identify two general methods depending on the particular nucleoside analogs. All the compounds were biologically evaluated in antiviral and anticancer assays and several showed improvement of activity compared to their parent nucleosides, as in the case of ddA, d4T, abacavir and acyclovir against HIV-1 and/or HIV-2. The biological results were supported by metabolism studies with carboxypeptidase Y monitored by (31)P NMR to investigate their bioactivation. This work further validates the phosphorodiamidate approach as a monophosphate prodrug motif with broad application in the antiviral and anticancer fields.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Nucleosídeos/química , Pró-Fármacos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-AtividadeRESUMO
7-Deazapurines are known to possess broad antiviral activity, however the 2'-C-methylguanosine analogue displays poor cell permeation and limited phosphorylation, thus is not an efficient inhibitor of hepatitis C virus (HCV) replication. We previously reported the 6-O-methyl entity as a prodrug moiety to increase liphophilicity of guanine nucleosides and the ProTide approach applied to 2'-C-methyl-6-O-methylguanosine has lead to potent HCV inhibitors now in clinical trials. In this Letter, we report the synthesis and biological evaluation of 2'-C-methyl-6-O-methyl-7-deaza guanosine and ProTide derivatives. In contrast to prior studies, removal of the N-7 of the nucleobase entirely negates anti-HCV activity compared to the 2'-C-methyl-6-O-methylguanosine analogues. To understand better this significant loss of activity, enzymatic assays and molecular modeling were carried out and suggested 2'-C-methyl-6-O-methyl-7-deaza guanosine and related ProTides do not act as efficient prodrugs of the free nucleotide, in marked contrast to the case of the parent guanine analogue.
Assuntos
Alanina/química , Amidas/farmacologia , Antivirais/farmacologia , Ésteres/farmacologia , Guanina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Ácidos Fosfóricos/farmacologia , Amidas/síntese química , Amidas/química , Antivirais/síntese química , Antivirais/química , Ésteres/síntese química , Ésteres/química , Guanina/síntese química , Guanina/química , Guanina/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Ácidos Fosfóricos/síntese química , Ácidos Fosfóricos/químicaRESUMO
Staphylococcus aureus is a Gram-positive pathogen that causes devastating disease and whose pathogenesis is dependent on interactions with host cell factors. Staphylococcal clumping factor A (ClfA) is a highly conserved fibrinogen (Fg)-binding protein and virulence factor that contributes to host tissue adhesion and initiation of infection. ClfA is being investigated as a possible component of a staphylococcal vaccine. We report the development of an Fg-binding assay that is specific for ClfA-mediated binding. Using the assay, we show that despite the presence of anti-ClfA antibodies, human sera from unvaccinated subjects are unable to prevent the binding of S. aureus to an Fg-coated surface. In contrast, antibodies elicited by a recombinant ClfA-containing vaccine were capable of blocking the ClfA-dependent binding of a diverse and clinically relevant collection of staphylococcal strains to Fg. These functional antibodies were also able to displace S. aureus already bound to Fg, suggesting that the ligand-binding activity of ClfA can be effectively neutralized through vaccination.
Assuntos
Aderência Bacteriana , Coagulase/imunologia , Fibrinogênio/metabolismo , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/sangue , Coagulase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/patogenicidadeRESUMO
We herein report phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven phosphorodiamidates are reported of two 6-O-alkyl 2'-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 µM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases.
Assuntos
Antivirais/síntese química , Guanosina/análogos & derivados , Guanosina/síntese química , Hepacivirus/efeitos dos fármacos , Compostos Organofosforados/síntese química , Pró-Fármacos/síntese química , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Catepsina A/metabolismo , Linhagem Celular , Estabilidade de Medicamentos , Guanosina/farmacocinética , Guanosina/farmacologia , Hepacivirus/genética , Humanos , Fígado/metabolismo , Masculino , Modelos Moleculares , Compostos Organofosforados/farmacocinética , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ratos , Ratos Sprague-Dawley , Soro , Relação Estrutura-AtividadeRESUMO
We have previously reported the power of combining a 5'-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2'-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , AMP Desaminase/metabolismo , Amidas/química , Amidas/metabolismo , Antivirais/química , Linhagem Celular Tumoral , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Guanosina Monofosfato/química , Guanosina Monofosfato/farmacologia , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Humanos , Hidrólise , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Nucleosídeos/farmacologia , Ácidos Fosfóricos/química , Ácidos Fosfóricos/metabolismo , Fosforilação , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.
Assuntos
Amidas/química , Antivirais/farmacologia , Antivirais/farmacocinética , Guanosina/farmacologia , Guanosina/farmacocinética , Hepacivirus/efeitos dos fármacos , Ácidos Fosfóricos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/farmacocinética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Guanosina/análogos & derivados , Guanosina/química , Humanos , Macaca fascicularis , Masculino , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Replicação Viral/efeitos dos fármacosRESUMO
We herein report a novel double pro-drug approach applied to the anti-HCV agent 2'-beta-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2'-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.
Assuntos
Antivirais/síntese química , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/síntese química , Amidas/química , Antivirais/química , Antivirais/toxicidade , Células Cultivadas , Desenho de Fármacos , Guanosina/análogos & derivados , Guanosina/síntese química , Guanosina/toxicidade , Guanosina Monofosfato/síntese química , Guanosina Monofosfato/química , Guanosina Monofosfato/farmacologia , Humanos , Ácidos Fosfóricos/química , Pró-Fármacos/química , Pró-Fármacos/toxicidadeRESUMO
Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.
Assuntos
Amidas/síntese química , Antivirais/síntese química , Guanosina/análogos & derivados , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Ácidos Fosfóricos/síntese química , Replicação Viral/efeitos dos fármacos , Trifosfato de Adenosina/análise , Amidas/química , Amidas/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Feminino , Guanosina/síntese química , Guanosina/química , Guanosina/farmacologia , Hepatite C/virologia , Humanos , Fígado/metabolismo , Fígado/virologia , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos ICR , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacologiaRESUMO
Two monoclonal antibodies (MAbs) were raised against the Candida albicans cell-surface glycoprotein Als3 using the N-terminal domain of the protein as the immunogen. ELISA was used to demonstrate the specificity of the MAbs for the Als3 fragment, but not for the corresponding N-terminal domain fragments from other proteins in the Als family. The anti-Als3 MAbs immunolabeled the surface of germ tubes from a diverse collection of wild-type C. albicans isolates, but did not label yeast cells, an als3Delta/als3Delta deletion mutant strain, nor isolates of other Candida species associated with human disease. Als3 was visualized readily in fresh and formalin-fixed, paraffin-embedded kidney tissue from a murine model of candidiasis. The anti-Als3 MAbs were also useful for immunogold electron microscopy and Western blotting. Both MAbs blocked C. albicans adhesion to vascular endothelial cells and buccal epithelial cells. These versatile MAbs are a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells.
Assuntos
Anticorpos Antifúngicos/imunologia , Anticorpos Monoclonais/imunologia , Candida albicans/fisiologia , Candidíase/microbiologia , Proteínas Fúngicas/imunologia , Interações Hospedeiro-Patógeno , Animais , Candida albicans/genética , Candida albicans/imunologia , Candidíase/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Enterococci are opportunistic pathogens known to cause numerous clinical infections and complications in humans. Adhesin-mediated binding to extracellular matrix (ECM) proteins of the host is thought to be a crucial step in the pathogenesis of these bacterial infections. Adhesin of collagen from Enterococcus faecalis (Ace) is a cell-wall anchored protein of E. faecalis that has been shown to be important for bacterial binding to the ECM. In this report, we characterize the conditions for Ace expression and demonstrate Ace binding to mammalian epithelial and endothelial cells as well as to collagens found in the ECM. To further characterize Ace expression and function, we report the generation of a panel of monoclonal antibodies (mAbs) directed against this important E. faecalis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance and flow cytometry, we have characterized this panel of mAbs which may prove to be not only beneficial in studies that address the precise biological role of adhesion of E. faecalis, but may also serve as beneficial therapeutic agents against E. faecalis infections.
Assuntos
Adesinas Bacterianas/metabolismo , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Colágeno/metabolismo , Enterococcus faecalis/imunologia , Adesinas Bacterianas/imunologia , Animais , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Enterococcus faecalis/metabolismo , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Staphylococcus epidermidis is an important opportunistic human pathogen that has recently emerged as a major cause of foreign-body infections. The most important stage contributing to the pathogenesis of this bacteria is the initial adherence to host tissue. SdrG is a cell-wall-anchored fibrinogen-binding adhesin of S. epidermidis that has been shown to be necessary for bacterial binding to fibrinogen-coated foreign bodies, such as catheters. Here we report the generation and characterization of a panel of monoclonal antibodies (MAbs) directed against this S. epidermidis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance, and flow cytometry, we have characterized a diverse array of MAbs that may prove to be beneficial in studies that address the precise biologic role of SdrG.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Fibrinogênio/metabolismo , Staphylococcus epidermidis/imunologia , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/imunologia , Staphylococcus epidermidis/genéticaRESUMO
A human donor-selected immunoglobulin G for intravenous injection (IGIV) product with elevated titers against the staphylococcal fibrinogen-binding MSCRAMM proteins ClfA and SdrG (INH-A21) was tested in vitro and in vivo. INH-A21 contained a significantly increased ability to inhibit the fibrinogen-binding activity of recombinant forms of both ClfA and SdrG. Evaluation of the opsonizing potential of INH-A21 was evaluated using fluorescently labeled bacteria; this assay indicated an increase in phagocytic activity compared to normal IGIV. The prophylactic efficacy of INH-A21 against an intraperitoneal challenge of methicillin-resistant Staphylococcus epidermidis (MRSE) was evaluated in a neonatal rat model. INH-A21 was also evaluated for prophylactic and therapeutic efficacy in a rabbit model of catheter-induced aortic valve infective endocarditis caused by either MRSE or methicillin-resistant Staphylococcus aureus (MRSA). Results from the in vivo models demonstrated potent prophylactic and therapeutic efficacy against both MRSE and MRSA. These data suggest that INH-A21 may be an important tool for the prevention and treatment of staphylococcal infections, especially in high-risk populations.
Assuntos
Anticorpos Antibacterianos/uso terapêutico , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Coagulase/imunologia , Imunoglobulina G/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/imunologia , Animais , Feminino , Humanos , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis.
Assuntos
Anticorpos Monoclonais/imunologia , Coagulase/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Monoclonais/sangue , Linhagem Celular , Humanos , CoelhosRESUMO
Staphylococcus capitis (S. capitis) has been implicated in a large proportion of coagulase-negative staphylococcal infections in very-low-birth-weight infants. To identify potential therapeutic targets, the S. capitis genome was probed for the presence of genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM). By using Southern blot analysis, an S. capitis gene, designated sdrX, that contained sequence motifs consistent with the Sdr family of MSCRAMM proteins was identified. By using monospecific antisera in Western blot and flow cytometry, SdrX was demonstrated to be expressed on the surface of S. capitis. Human collagen type VI was found to bind both the recombinant A domain of SdrX and viable S. capitis expressing SdrX. SdrX is the first collagen-binding Sdr protein described and is the first MSCRAMM protein identified in S. capitis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Colágeno/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Staphylococcus/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Ácido Aspártico , Proteínas de Bactérias/genética , Clonagem Molecular , Humanos , Recém-Nascido , Proteínas de Membrana/genética , Dados de Sequência Molecular , Serina , Staphylococcus/genética , Staphylococcus/crescimento & desenvolvimentoRESUMO
Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Ativação do Complemento/efeitos dos fármacos , Complemento C3/metabolismo , Staphylococcus aureus/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Opsonizantes/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Coagulase/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Aderência Bacteriana , Coagulase/genética , Humanos , Hibridomas , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/mortalidade , Staphylococcus aureus/patogenicidade , Ressonância de Plasmônio de SuperfícieRESUMO
SA-IGIV is a human polyclonal immunoglobulin containing elevated levels of antibodies specific for the fibrinogen-binding MSCRAMM protein clumping factor A (ClfA). In vitro, SA-IGIV specifically recognized ClfA that was expressed on the surface of Staphylococcus aureus and inhibited bacterial adherence to immobilized human fibrinogen by >95%. Moreover, SA-IGIV efficiently opsonized ClfA-coated fluorescent beads and facilitated phagocytosis by human polymorphonuclear leukocytes. To determine its potential therapeutic efficacy, SA-IGIV was evaluated in combination with vancomycin in a rabbit model of catheter-induced aortic valve infective endocarditis (IE) caused by methicillin-resistant S. aureus (MRSA). The combination therapy was more effective than vancomycin alone in sterilizing all valvular vegetations when used therapeutically during early (12-h) IE. The combination therapy resulted in clearance of bacteremia that was significantly faster than that of vancomycin alone in animals with well-established (24-h) IE. Therefore, in both early and well-established MRSA IE, the addition of SA-IGIV to a standard antibiotic regimen (vancomycin) increased bacterial clearance from the bloodstream and/or vegetations.