RESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked â¼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.
Assuntos
Aminofenóis , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Simulação de Acoplamento Molecular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Aminofenóis/farmacologia , Aminofenóis/química , Aminofenóis/uso terapêutico , Descoberta de Drogas , Microscopia Crioeletrônica , Quinolonas/farmacologia , Quinolonas/química , Quinolonas/uso terapêutico , Sítio Alostérico/efeitos dos fármacos , Animais , LigantesRESUMO
Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Histonas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Vírus da Encefalomiocardite/fisiologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Interferon beta/farmacologia , Interferon gama/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Mutação , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Interferência de RNA , DNA Polimerase Dirigida por RNA , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Transcriptoma/efeitos dos fármacos , Ubiquitina-Proteína Ligases/genéticaRESUMO
Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.
Assuntos
Antígenos de Protozoários/química , Sistema do Grupo Sanguíneo Duffy/química , Eritrócitos/química , Plasmodium vivax/química , Proteínas de Protozoários/química , Receptores de Superfície Celular/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Linhagem Celular , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Humanos , Evasão da Resposta Imune , Malária Vivax/genética , Malária Vivax/imunologia , Plasmodium vivax/imunologia , Plasmodium vivax/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Relação Estrutura-AtividadeRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked ~155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.
RESUMO
BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Fezes , Doenças Inflamatórias Intestinais , Mucosa Intestinal , Humanos , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/genética , Clostridium perfringens/patogenicidade , Criança , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Doenças Inflamatórias Intestinais/microbiologia , Toxinas Bacterianas/genética , Fezes/microbiologia , Feminino , Masculino , Adolescente , Biópsia , Infecções por Clostridium/microbiologia , Proteínas HemolisinasRESUMO
Urinary tract infections (UTIs) cause a substantial health care burden. UTIs (i) are most often caused by uropathogenic Escherichia coli (UPEC), (ii) primarily affect otherwise healthy females (50% of women will have a UTI), (iii) are associated with significant morbidity and economic impact, (iv) can become chronic, and (v) are highly recurrent. A history of UTI is a significant risk factor for a recurrent UTI (rUTI). In otherwise healthy women, an acute UTI leads to a 25 to 50% chance of rUTI within months of the initial infection. Interestingly, rUTIs are commonly caused by the same strain of E. coli that led to the initial infection, arguing that there exist host-associated reservoirs, like the gastrointestinal tract and underlying bladder tissue, that can seed rUTIs. Additionally, catheter-associated UTIs (CAUTI), caused by Enterococcus and Staphylococcus as well as UPEC, represent a major health care concern. The host's response of depositing fibrinogen at the site of infection has been found to be critical to establishing CAUTI. The Drug Resistance Index, an evaluation of antibiotic resistance, indicates that UTIs have become increasingly difficult to treat since the mid-2000s. Thus, UTIs are a "canary in the coal mine," warning of the possibility of a return to the preantibiotic era, where some common infections are untreatable with available antibiotics. Numerous alternative strategies for both the prevention and treatment of UTIs are being pursued, with a focus on the development of vaccines and small-molecule inhibitors targeting virulence factors, in the hopes of reducing the burden of urogenital tract infections in an antibiotic-sparing manner.
Assuntos
Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Antibacterianos/uso terapêutico , Infecções Relacionadas a Cateter/microbiologia , Farmacorresistência Bacteriana , Enterococcus/patogenicidade , Infecções por Escherichia coli/tratamento farmacológico , Feminino , Humanos , Staphylococcus/patogenicidade , Bexiga Urinária/microbiologia , Sistema Urinário/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacosRESUMO
Chaperone-usher pathway pili are extracellular proteinaceous fibres ubiquitously found on Gram-negative bacteria, and mediate host-pathogen interactions and biofilm formation critical in pathogenesis in numerous human diseases1. During pilus assembly, an outer membrane macromolecular machine called the usher catalyses pilus biogenesis from the individual subunits that are delivered as chaperone-subunit complexes in the periplasm. The usher orchestrates pilus assembly using all five functional domains: a 24-stranded transmembrane ß-barrel translocation domain, a ß-sandwich plug domain, an amino-terminal periplasmic domain and two carboxy-terminal periplasmic domains (CTD1 and CTD2)2-6. Despite extensive structural and functional characterization, the mechanism by which the usher is activated to initiate pilus biogenesis is unknown. Here, we present the crystal structure of the full-length PapC usher from Escherichia coli in complex with its cognate PapDG chaperone-subunit complex in a pre-activation state, elucidating molecular details of how the usher is specifically engaged by allosteric interactions with its substrate preceding activation and how the usher facilitates the transfer of subunits from the amino-terminal periplasmic domain to the CTDs during pilus assembly. This work elucidates the intricate workings of a molecular machine that catalyses chaperone-usher pathway pilus assembly and opens the door for the development of potent inhibitors to block pilus biogenesis.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Fímbrias Bacterianas/química , Porinas/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Proteínas Periplásmicas/genética , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
BST-2 (also known as tetherin, CD317, or HM1.24) was first described as a potent interferon-inducible host antiviral factor nearly five years ago. Since that time, numerous reports have been published regarding the antiviral activity and immunological properties of this protein. BST-2 blocks viral replication by inhibiting enveloped virus budding from the surface of infected cells. To counteract this, most viruses have developed strategies to antagonize BST-2, each employing a unique mechanism. In this review, we summarize the antiviral function, structural biology and immunobiology of BST-2. Taken together, our current understanding of BST-2 suggests potential avenues as well as challenges to exploiting its action in the development of broad spectrum antiviral treatments.