RESUMEN
We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and a therapeutic drug for the treatment of COVID-19 given intraperitoneally in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research, but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.
Asunto(s)
Antivirales , Benzotiazoles , Tratamiento Farmacológico de COVID-19 , Oligopéptidos , SARS-CoV-2 , Inhibidores de Serina Proteinasa , Replicación Viral , Animales , Femenino , Humanos , Ratones , Antivirales/farmacología , Chlorocebus aethiops , COVID-19/virología , Modelos Animales de Enfermedad , Pulmón/virología , Pulmón/patología , Pulmón/efectos de los fármacos , Peptidomiméticos/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Inhibidores de Serina Proteinasa/uso terapéutico , Células Vero , Replicación Viral/efectos de los fármacos , Oligopéptidos/farmacología , Benzotiazoles/farmacologíaRESUMEN
Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.
Asunto(s)
Neoplasias Colorrectales , Transducción de Señal , Humanos , Cetuximab/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Resistencia a Antineoplásicos/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Inhibidores de Proteasas/farmacología , Péptido Hidrolasas/metabolismo , Línea Celular Tumoral , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacologíaRESUMEN
The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.
Asunto(s)
Benzotiazoles/farmacología , Tratamiento Farmacológico de COVID-19 , Oligopéptidos/farmacología , SARS-CoV-2/efectos de los fármacos , Serina Endopeptidasas/genética , Animales , Benzamidinas/química , Benzotiazoles/farmacocinética , COVID-19/genética , COVID-19/virología , Línea Celular , Diseño de Fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Ésteres/química , Guanidinas/química , Humanos , Pulmón/efectos de los fármacos , Pulmón/virología , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Oligopéptidos/farmacocinética , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/efectos de los fármacos , Serina Endopeptidasas/ultraestructura , Bibliotecas de Moléculas Pequeñas/farmacología , Especificidad por Sustrato/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.
Asunto(s)
Adhesinas de Escherichia coli/metabolismo , Desmogleína 2/metabolismo , Infecciones por Escherichia coli/microbiología , Escherichia coli/patogenicidad , Proteínas Fimbrias/metabolismo , Pielonefritis/microbiología , Adhesinas de Escherichia coli/genética , Animales , Desmogleína 2/genética , Epitelio/microbiología , Escherichia coli/genética , Femenino , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Vejiga Urinaria/microbiología , VirulenciaRESUMEN
Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.
Asunto(s)
Proteínas Fimbrias/antagonistas & inhibidores , Intestinos/efectos de los fármacos , Intestinos/microbiología , Manósidos/farmacología , Ácidos Ftálicos/farmacología , Infecciones Urinarias/prevención & control , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/aislamiento & purificación , Adhesinas de Escherichia coli/metabolismo , Secuencia de Aminoácidos , Animales , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Heces/microbiología , Femenino , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/clasificación , Fimbrias Bacterianas/efectos de los fármacos , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Intestinos/citología , Manósidos/uso terapéutico , Ratones , Modelos Moleculares , Ácidos Ftálicos/uso terapéutico , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/microbiología , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/clasificación , Escherichia coli Uropatógena/genéticaRESUMEN
Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone-usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29ß-NAc, is a biphenyl N-acetyl-ß-galactosaminoside with a Ki of â¼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate-lectin interactions. 29ß-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29ß-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29ß-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29ß-NAc-inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.
Asunto(s)
Adhesinas de Escherichia coli/química , Antibacterianos/química , Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Infecciones Urinarias/microbiología , Adhesinas de Escherichia coli/metabolismo , Animales , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Femenino , Galactósidos/síntesis química , Galactósidos/química , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/microbiología , Ligandos , Ratones Endogámicos C3H , Simulación del Acoplamiento Molecular , Imitación Molecular , Infecciones Urinarias/tratamiento farmacológico , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/patogenicidadRESUMEN
Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Imidazoles/farmacología , Receptor Smoothened/antagonistas & inhibidores , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzamidas/síntesis química , Benzamidas/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Imidazoles/síntesis química , Imidazoles/química , Ratones , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Receptor Smoothened/metabolismo , Relación Estructura-Actividad , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Voltage-dependent anion channel-1 (VDAC1) is a highly regulated ß-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue.
Asunto(s)
Colesterol/química , Canal Aniónico 1 Dependiente del Voltaje/química , Marcadores de Afinidad , Animales , Sitios de Unión , Ratones , Resonancia Magnética Nuclear Biomolecular , Canal Aniónico 1 Dependiente del Voltaje/genéticaRESUMEN
Identifying sites of protein-ligand interaction is important for structure-based drug discovery and understanding protein structure-function relationships. Mass spectrometry (MS) has emerged as a useful tool for identifying residues covalently modified by ligands. Current methods use database searches that are dependent on acquiring interpretable fragmentation spectra (MS2) of peptide-ligand adducts. This is problematic for identifying sites of hydrophobic ligand incorporation in integral membrane proteins (IMPs), where poor aqueous solubility and ionization of peptide-ligand adducts and collision-induced adduct loss hinder the acquisition of quality MS2 spectra. To address these issues, we developed a fast ligand identification (FLI) tag that can be attached to any alkyne-containing ligand via Cu(I)-catalyzed cycloaddition. The FLI tag adds charge to increase solubility and ionization, and utilizes stable isotope labeling for MS1 level identification of hydrophobic peptide-ligand adducts. The FLI tag was coupled to an alkyne-containing neurosteroid photolabeling reagent and used to identify peptide-steroid adducts in MS1 spectra via the stable heavy isotope pair. Peptide-steroid adducts were not identified in MS2-based database searches because collision-induced adduct loss was the dominant feature of collision-induced dissociation (CID) fragmentation, but targeted analysis of MS1 pairs using electron transfer dissociation (ETD) markedly reduced adduct loss. Using the FLI tag and ETD, we identified Glu73 as the site of photoincorporation of our neurosteroid ligand in the IMP, mouse voltage-dependent anion channel-1 (mVDAC1), and top-down MS confirmed a single site of photolabeling.
Asunto(s)
Ligandos , Péptidos/química , Espectrometría de Masas en Tándem , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Alquinos/química , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Química Clic , Interacciones Hidrofóbicas e Hidrofílicas , Marcaje Isotópico , Ratones , Péptidos/metabolismo , Solubilidad , Rayos Ultravioleta , Canal Aniónico 1 Dependiente del Voltaje/químicaRESUMEN
Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA.
Asunto(s)
Antineoplásicos/síntesis química , Benzamidinas/síntesis química , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Serina Endopeptidasas/química , Amidinas/síntesis química , Antineoplásicos/farmacología , Arginina/química , Benzamidinas/farmacología , Dominio Catalítico , Diseño de Fármacos , Pruebas de Enzimas , Guanidinas/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/química , Peptidomiméticos/química , Fenilalanina/análogos & derivados , Fenilalanina/síntesis química , Piperazinas/síntesis química , Inhibidores de Proteasas/farmacología , Proteínas Recombinantes/química , Relación Estructura-Actividad , Urea/análogos & derivados , Urea/químicaRESUMEN
BACKGROUND: Escherichia coli O25b:H4-ST131 represents a predominant clone of multidrug-resistant uropathogens currently circulating worldwide in hospitals and the community. Urinary tract infections (UTIs) caused by E. coli ST131 are typically associated with limited treatment options and are often recurrent. METHODS: Using established mouse models of acute and chronic UTI, we mapped the pathogenic trajectory of the reference E. coli ST131 UTI isolate, strain EC958. RESULTS: We demonstrated that E. coli EC958 can invade bladder epithelial cells and form intracellular bacterial communities early during acute UTI. Moreover, E. coli EC958 persisted in the bladder and established chronic UTI. Prophylactic antibiotic administration failed to prevent E. coli EC958-mediated UTI. However, 1 oral dose of a small-molecular-weight compound that inhibits FimH, the type 1 fimbriae adhesin, significantly reduced bacterial colonization of the bladder and prevented acute UTI. Treatment of chronically infected mice with the same FimH inhibitor lowered their bladder bacterial burden by >1000-fold. CONCLUSIONS: In this study, we provide novel insight into the pathogenic mechanisms used by the globally disseminated E. coli ST131 clone during acute and chronic UTI and establish the potential of FimH inhibitors as an alternative treatment against multidrug-resistant E. coli.
Asunto(s)
Cistitis/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple , Proteínas Fimbrias/antagonistas & inhibidores , Escherichia coli Uropatógena/aislamiento & purificación , Enfermedad Aguda , Adhesinas de Escherichia coli , Administración Oral , Animales , Antibacterianos/uso terapéutico , Enfermedad Crónica , Cistitis/microbiología , Cistitis/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Fimbrias Bacterianas/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C3H , Vejiga Urinaria/microbiologíaRESUMEN
FmlH, a bacterial adhesin of uropathogenic Escherichia coli (UPEC), has been shown to provide a fitness advantage in colonizing the bladder during chronic urinary tract infections (UTIs). Previously reported ortho-biphenyl glycosides based on ßGal and ßGalNAc have excellent binding affinity to FmlH and potently block binding to its natural carbohydrate receptor, but they lack oral bioavailability. In this paper, we outline studies where we have optimized compounds for improved pharmacokinetics, leading to the discovery of novel analogues with good oral bioavailability. We synthesized galactosides with the anomeric O-linker replaced with more stable S- and C-linked linkers. We also investigated modifications to the GalNAc sugar and modifications to the biphenyl aglycone. We identified GalNAc 69 with an IC50 of 0.19 µM against FmlH and 53% oral bioavailability in mice. We also obtained a FimlH-bound X-ray structure of lead compound 69 (AM4085) which has potential as a new antivirulence therapeutic for UTIs.
Asunto(s)
Infecciones por Escherichia coli , Infecciones Urinarias , Escherichia coli Uropatógena , Ratones , Animales , Lectinas , Adhesinas de Escherichia coli/química , Infecciones Urinarias/tratamiento farmacológico , Compuestos de Bifenilo/química , Escherichia coli Uropatógena/metabolismo , Infecciones por Escherichia coli/tratamiento farmacológicoRESUMEN
The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7â µM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.
Asunto(s)
Cisplatino , Neoplasias Testiculares , Humanos , Masculino , Cisplatino/farmacología , ADN/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/química , Endonucleasas/metabolismo , Péptidos/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/química , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , FemeninoRESUMEN
Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.
Asunto(s)
Serina Proteasas , Inhibidores de Serina Proteinasa , Animales , Ratones , Serina Proteasas/metabolismo , Relación Estructura-Actividad , Inhibidores de Serina Proteinasa/química , Conformación Molecular , PéptidosRESUMEN
We have developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases including transmembrane protease serine 2 (TMPRSS2), matriptase and hepsin. TMPRSS2 is a membrane associated protease which is highly expressed in the upper and lower respiratory tract and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell receptor binding, entry, replication, and dissemination of new virion particles. We have previously shown that compound MM3122 exhibited sub nanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell-viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Further, we have evaluated MM3122 in a mouse model of COVID-19 and have demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion, and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in pro-inflammatory cytokines and chemokines production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2 infected mice. Therefore, MM3122 is a promising lead candidate small molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host-cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and therapeutic drug for the treatment of COVID-19 in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.
RESUMEN
Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design.
Asunto(s)
Serina Endopeptidasas , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Humanos , Diseño de Fármacos , Aminoácidos/química , Aminoácidos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Animales , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/antagonistas & inhibidoresRESUMEN
Urinary tract infections (UTI), primarily caused by uropathogenic Escherichia coli (UPEC), are one of the leading bacterial infections due to their high frequency and rate of recurrence. Both type 1 pilus adhesive organelles (fim) and the QseC sensor kinase have been implicated in UPEC virulence during UTI and have been individually reported to be promising drug targets. Deletion of qseC leads to pleiotropic effects due to unregulated activation of the cognate response regulator QseB, influencing conserved metabolic processes and diminishing expression of virulence genes, including type 1 pili. Here, we discern the type 1 pilus-dependent and -independent effects that contribute to the virulence attenuation of a UPEC qseC deletion mutant in a murine model of experimental UTI. We show that although a ΔqseC mutant restored for type 1 pilus expression regains the ability to colonize the host and initiate acute infection up to 16 h postinfection, it is rapidly outcompeted during acute infection when coinoculated with a wild-type strain. As a result, this strain has a diminished capacity to establish chronic infection. A prophylactic oral dose of a FimH small-molecular-weight antagonist (ZFH-02056) further reduced the ability of the qseC mutant to establish chronic infection. Thus, loss of QseC significantly enhances the efficacy of ZFH-02056. Collectively, our work indicates that type 1 pili and QseC become critical in different infection stages, and that dual targeting of these factors has an additive effect on ablating UPEC virulence.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/metabolismo , Animales , Proteínas de Escherichia coli/genética , Femenino , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C3H , Mutación , Vejiga Urinaria/microbiología , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/patogenicidad , VirulenciaRESUMEN
Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial urinary tract infections (UTIs) and pose significant clinical challenges. These infections are polymicrobial in nature and are often associated with multidrug-resistant pathogens, including uropathogenic Escherichia coli (UPEC). Urinary catheterization elicits major histological and immunological alterations in the bladder that can favor microbial colonization and dissemination in the urinary tract. We report that these biological perturbations impact UPEC pathogenesis and that bacterial reservoirs established during a previous UPEC infection, in which bacteriuria had resolved, can serve as a nidus for subsequent urinary catheter colonization. Mannosides, small molecule inhibitors of the type 1 pilus adhesin, FimH, provided significant protection against UPEC CAUTI by preventing bacterial invasion and shifting the UPEC niche primarily to the extracellular milieu and on the foreign body. By doing so, mannosides potentiated the action of trimethoprim-sulfamethoxazole in the prevention and treatment of CAUTI. In this study, we provide novel insights into UPEC pathogenesis in the context of urinary catheterization, and demonstrate the efficacy of novel therapies that target critical mechanisms for this infection. Thus, we establish a proof-of-principle for the development of mannosides to prevent and eventually treat these infections in the face of rising antibiotic-resistant uropathogens.
Asunto(s)
Infecciones Relacionadas con Catéteres/tratamiento farmacológico , Infección Hospitalaria/tratamiento farmacológico , Infecciones por Escherichia coli/tratamiento farmacológico , Manósidos/farmacología , Combinación Trimetoprim y Sulfametoxazol/farmacología , Infecciones Urinarias/tratamiento farmacológico , Escherichia coli Uropatógena/efectos de los fármacos , Adhesinas de Escherichia coli/genética , Animales , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Infecciones Relacionadas con Catéteres/microbiología , Infección Hospitalaria/microbiología , Quimioterapia Combinada , Infecciones por Escherichia coli/microbiología , Femenino , Proteínas Fimbrias/deficiencia , Proteínas Fimbrias/genética , Eliminación de Gen , Manósidos/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Combinación Trimetoprim y Sulfametoxazol/uso terapéutico , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/microbiología , Catéteres Urinarios/microbiología , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/crecimiento & desarrollo , Escherichia coli Uropatógena/patogenicidadRESUMEN
Cell-based subset screening of compounds using a Gli transcription factor reporter cell assay and shh stimulated cell differentiation assay identified a series of bisamide compounds as hedgehog pathway inhibitors with good potency. Using a ligand-based optimization strategy, heteroaryl groups were utilized as conformationally restricted amide isosteres replacing one of the amides which significantly increased their potency against SMO and the hedgehog pathway while decreasing activity against p38α kinase. We report herein the identification of advanced lead compounds such as imidazole 11c and 11f encompassing good p38α selectivity, low nanomolar potency in both cell assays, excellent physiochemical properties and in vivo pharmacokinetics.
Asunto(s)
Amidas/química , Proteínas Hedgehog/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Amidas/farmacología , Animales , Evaluación Preclínica de Medicamentos , Ratones , Estructura Molecular , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-ActividadRESUMEN
The current treatments for lymphatic filariasis and onchocerciasis do not effectively kill the adult parasitic nematodes, allowing these chronic and debilitating diseases to persist in millions of people. Thus, the discovery of new drugs with macrofilaricidal potential to treat these filarial diseases is critical. To facilitate this need, we first investigated the effects of three aspartyl protease inhibitors (APIs) that are FDA-approved as HIV antiretroviral drugs on the adult filarial nematode, Brugia malayi and the endosymbiotic bacteria, Wolbachia. From the three hits, nelfinavir had the best potency with an IC50 value of 7.78 µM, followed by ritonavir and lopinavir with IC50 values of 14.3 µM and 16.9 µM, respectively. The three APIs have a direct effect on killing adult B. malayi after 6 days of exposure in vitro and did not affect the Wolbachia titers. Sequence conservation and stage-specific gene expression analysis identified Bm8660 as the most likely primary aspartic protease target for these drug(s). Immunolocalization using antibodies raised against the Bm8660 ortholog of Onchocerca volvulus showed it is strongly expressed in female B. malayi, especially in metabolically active tissues such as lateral and dorsal/ventral chords, hypodermis, and uterus tissue. Global transcriptional response analysis using adult female B. pahangi treated with APIs identified four additional aspartic proteases differentially regulated by the three effective drugs, as well as significant enrichment of various pathways including ubiquitin mediated proteolysis, protein kinases, and MAPK/AMPK/FoxO signaling. In vitro testing against the adult gastro-intestinal nematode Trichuris muris suggested broad-spectrum potential for these APIs. This study suggests that APIs may serve as new leads to be further explored for drug discovery to treat parasitic nematode infections.