RESUMEN
Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.
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Descubrimiento de Drogas , SARS-CoV-2/fisiología , Tropismo Viral , Internalización del Virus , Replicación Viral , COVID-19/virología , Catepsinas , Línea Celular , Desarrollo de Medicamentos , Endocitosis , Furina , Humanos , SARS-CoV-2/efectos de los fármacos , Serina Endopeptidasas , Tratamiento Farmacológico de COVID-19RESUMEN
A property-focused optimization strategy was employed to modify the carboxylic acid head group of a class of EP4 agonists in order to minimize its absorption upon oral administration. The resulting oxalic acid monohydrazide-derived carboxylate isostere demonstrated utility as a class of prodrug showing colon-targeted delivery of parent agonist 2, with minimal exposure observed in the plasma. Oral administration of NXT-10796 demonstrated tissue specific activation of the EP4 receptor through modulation of immune genes in the colon, without modulation of EP4 driven biomarkers in the plasma compartment. Although further in depth understanding of the conversion of NXT-10796 is required for further assessment of the developability of this series of prodrugs, using NXT-10796 as a tool molecule has allowed us to confirm that tissue-specific modulation of an EP4-modulated gene signature is possible, which allows for further evaluation of this therapeutic modality in rodent models of human disease.
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Enfermedades Inflamatorias del Intestino , Profármacos , Humanos , Profármacos/farmacología , Profármacos/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Colon , Subtipo EP4 de Receptores de Prostaglandina E/agonistasRESUMEN
Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.
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Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Clostridioides difficile/enzimología , Glucosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/genética , Anticuerpos ampliamente neutralizantes , Células CHO , Células CACO-2 , Chlorocebus aethiops , Proteoglicanos Tipo Condroitín Sulfato/genética , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Cricetinae , Cricetulus , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Unión Proteica , Dominios ProteicosRESUMEN
Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab.
Asunto(s)
Antibacterianos/efectos adversos , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Antitoxinas/farmacología , Infecciones por Clostridium/tratamiento farmacológico , Vancomicina/efectos adversos , Animales , Antibacterianos/administración & dosificación , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/biosíntesis , Anticuerpos ampliamente neutralizantes , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/crecimiento & desarrollo , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/inmunología , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/mortalidad , Convalecencia , Cricetulus , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Enterotoxinas/antagonistas & inhibidores , Enterotoxinas/biosíntesis , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Análisis de Supervivencia , Vancomicina/administración & dosificaciónRESUMEN
The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a ß-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/química , Toxinas Bacterianas/inmunología , Clostridioides difficile/inmunología , Epítopos/inmunología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales , Anticuerpos Neutralizantes/química , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Sitios de Unión , Anticuerpos ampliamente neutralizantes , Clostridioides difficile/química , Clostridioides difficile/genética , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos/química , Epítopos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antitoxinas/inmunología , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Clostridioides difficile/inmunología , Enterocolitis Seudomembranosa/prevención & control , Enterotoxinas/inmunología , Animales , Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Antitoxinas/uso terapéutico , Chlorocebus aethiops , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/inmunología , Ratones , Ratones Endogámicos C57BL , Mutación , Receptores de IgG/inmunología , Recurrencia , Células VeroRESUMEN
Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.
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Antibacterianos/farmacología , Anticuerpos Monoclonales/farmacología , Clostridioides difficile/efectos de los fármacos , Proteínas Bacterianas/genética , Línea Celular , Clostridioides difficile/inmunología , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/microbiología , Epítopos/inmunología , Femenino , Humanos , MasculinoRESUMEN
The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.
Asunto(s)
Antibacterianos , Inhibidores de beta-Lactamasas , Humanos , Inhibidores de beta-Lactamasas/farmacología , Inhibidores de beta-Lactamasas/uso terapéutico , Inhibidores de beta-Lactamasas/química , Antibacterianos/química , Imipenem/farmacología , beta-Lactamasas , Bacterias Gramnegativas , Pruebas de Sensibilidad MicrobianaRESUMEN
Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Despite continued efforts to understand the pathophysiology of sepsis, no effective therapies are currently available. While singular components of the aberrant immune response have been investigated, comprehensive studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and concomitant changes in cytokines and metabolites in patients with sepsis. Our findings identify differentially expressed mature and immature neutrophil subsets in patients with sepsis. These subsets correlate with various proteins, metabolites, and lipids, including pentraxin-3, angiopoietin-2, and lysophosphatidylcholines, in patients with sepsis. These results enabled the construction of a statistical model based on weighted multi-omics linear regression analysis for sepsis biomarker identification. These findings could help inform early patient stratification and treatment options, and facilitate further mechanistic studies targeting the trifecta of surface marker expression, cytokines, and metabolites.
RESUMEN
Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (µVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DµVS), that integrates a µVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DµVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DµVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.
RESUMEN
The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Compuestos de Bifenilo/farmacología , Depsipéptidos/farmacología , Glicopéptidos/farmacología , Glicósidos/farmacología , Lipopéptidos/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Oligopéptidos/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , beta-Lactamas/farmacología , Animales , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Compuestos de Bifenilo/síntesis química , Depsipéptidos/aislamiento & purificación , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Glicopéptidos/síntesis química , Glicopéptidos/aislamiento & purificación , Glicósidos/aislamiento & purificación , Humanos , Lipopéptidos/aislamiento & purificación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Familia de Multigenes , Oligopéptidos/síntesis química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Infecciones Estafilocócicas/microbiología , Resistencia betalactámica/efectos de los fármacos , Resistencia betalactámica/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
BACKGROUND: Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system. METHODS: Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools. RESULTS: An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts. CONCLUSIONS: These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.
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Quimiocina CCL11/metabolismo , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Factor 2 Relacionado con NF-E2/genética , ARN Interferente Pequeño/genéticaRESUMEN
The host response to SARS-CoV-2, the virus that causes COVID-19, is highly heterogeneous, ranging from mild/asymptomatic to severe. The moderate to severe forms of COVID-19 often require hospitalization, are associated with a high rate of mortality, and appear to be caused by an inappropriately exaggerated inflammatory response to the virus. Emerging data confirm the involvement of both innate and adaptive immune pathways both in protection from SARS-CoV-2, and in driving the pathology of severe COVID-19. In particular, innate immune cells including neutrophils appear to be key players in the inflammation that causes the vicious cycle of damage and inflammation that underlies the symptomatology of severe COVID-19. Several recent studies support a link between damage and inflammation, with damage-associated molecular patterns (DAMPs) playing a key role in the pathology of severe COVID-19. In this review, we put into perspective the role of DAMPs and of components of the DAMP-signaling cascade, including Siglecs and their cognate ligands CD24 and CD52, in COVID-19. Further, we review clinical data on proposed therapeutics targeting DAMP pathways to treat SARS-CoV-2 infection and the regulation of these signaling cascades in COVID-19. We also discuss the potential impact of DAMP-mediated inflammation in other indications related to COVID-19, such as ARDS, endothelial dysfunction, hypercoagulation, and sepsis.
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Alarminas/metabolismo , COVID-19/metabolismo , COVID-19/patología , Mediadores de Inflamación/metabolismo , Inflamación/metabolismo , SARS-CoV-2 , Humanos , Inmunidad Innata , Inflamación/patologíaRESUMEN
With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.
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Infecciones Bacterianas , Inhibidores de beta-Lactamasas , Animales , Ratones , Inhibidores de beta-Lactamasas/farmacología , Inhibidores de beta-Lactamasas/uso terapéutico , Inhibidores de beta-Lactamasas/química , Imipenem/farmacología , Imipenem/uso terapéutico , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/química , Pruebas de Sensibilidad MicrobianaRESUMEN
We disclose herein our preliminary SAR study on the identification of substituted benzothiophene derivatives as PGE(2) subtype 4 receptor antagonists. A potent EP(4) antagonist 6a (K(i)=1.4nM with 10% HSA) was identified. Furthermore, we found that an acidic group was not essential for the EP(4) antagonizing activity in the series and neutral replacements were identified. This opens a new direction for future EP(4) antagonist design.
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Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Tiofenos/química , Tiofenos/farmacología , Línea Celular , Humanos , Unión Proteica , Relación Estructura-Actividad , Tiofenos/síntesis químicaRESUMEN
A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.
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Indoles/química , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Animales , Artritis/inducido químicamente , Artritis/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Indoles/farmacocinética , Indoles/uso terapéutico , Ligandos , Ratas , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Relación Estructura-ActividadRESUMEN
Two new series of EP(4) antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.
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Amidas/química , Indoles/química , Naftalenos/química , Quinolinas/química , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Compuestos de Sulfonilurea/química , Amidas/síntesis química , Amidas/farmacocinética , Animales , Perros , Evaluación Preclínica de Medicamentos , Humanos , Indoles/síntesis química , Indoles/farmacocinética , Ratas , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Relación Estructura-Actividad , Compuestos de Sulfonilurea/síntesis química , Compuestos de Sulfonilurea/farmacocinéticaRESUMEN
Emerging evidence in clinical and preclinical studies indicates that success of immunotherapies can be impacted by the state of the microbiome. Understanding the role of the microbiome during immune-targeted interventions could help us understand heterogeneity of treatment success, predict outcomes, and develop additional strategies to improve efficacy. In this review, we discuss key studies that reveal reciprocal interactions between the microbiome, the immune system, and the outcome of immune interventions. We focus on cancer immune checkpoint inhibitor treatment and vaccination as two crucial therapeutic areas with strong potential for immunomodulation by the microbiota. By juxtaposing studies across both therapeutic areas, we highlight three factors prominently involved in microbial immunomodulation: short-chain fatty acids, microbe-associate molecular patterns (MAMPs), and inflammatory cytokines. Continued interrogation of these models and pathways may reveal critical mechanistic synergies between the microbiome and the immune system, resulting in novel approaches designed to influence the efficacy of immune-targeted interventions.
Asunto(s)
Microbioma Gastrointestinal/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunomodulación/efectos de los fármacos , Inmunoterapia , Neoplasias , Humanos , Neoplasias/inmunología , Neoplasias/microbiología , Neoplasias/terapiaRESUMEN
The discovery of a highly potent and selective EP(4) antagonist MF-766 is discussed. This N-benzyl indole derivative exhibits good pharmacokinetic profile and unprecedented in vivo potency in the rat AIA model.
Asunto(s)
Benzoatos/farmacología , Indoles/farmacología , Dolor/tratamiento farmacológico , Receptores de Prostaglandina E/antagonistas & inhibidores , Animales , Artritis/tratamiento farmacológico , Benzoatos/química , Benzoatos/uso terapéutico , Células Cultivadas , Perros , Descubrimiento de Drogas , Estabilidad de Medicamentos , Hepatocitos , Humanos , Indoles/química , Indoles/uso terapéutico , Inflamación/tratamiento farmacológico , Farmacocinética , Ratas , Subtipo EP4 de Receptores de Prostaglandina E , Relación Estructura-ActividadRESUMEN
Interleukin 13 (IL-13) is considered to be a key driver of the development of airway allergic inflammation and remodeling leading to airway hyperresponsiveness (AHR). How precisely IL-13 leads to the development of airway inflammation, AHR, and mucus production is not fully understood. In order to identify key mediators downstream of IL-13, we administered adenovirus IL-13 to specifically induce IL-13-dependent inflammation in the lungs of mice. This approach was shown to induce cardinal features of lung disease, specifically airway inflammation, elevated cytokines, AHR, and mucus secretion. Notably, the model is resistant to corticosteroid treatment and is characterized by marked neutrophilia, two hallmarks of more severe forms of asthma. To identify IL-13-dependent mediators, we performed a limited-scale two-dimensional SDS-PAGE proteomic analysis and identified proteins significantly modulated in this model. Intriguingly, several identified proteins were unique to this model, whereas others correlated with those modulated in a mouse ovalbumin-induced pulmonary inflammation model. We corroborated this approach by illustrating that proteomic analysis can identify known pathways/mediators downstream of IL-13. Thus, we have characterized a murine adenovirus IL-13 lung model that recapitulates specific disease traits observed in human asthma, and have exploited this model to identify effectors downstream of IL-13. Collectively, these findings will enable a broader appreciation of IL-13 and its impact on disease pathways in the lung.