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1.
Cell ; 182(2): 345-356.e16, 2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32589945

RESUMEN

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.


Asunto(s)
Toxinas Bacterianas/metabolismo , Clostridium sordellii/metabolismo , Semaforinas/metabolismo , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidad , Sitios de Unión , Sistemas CRISPR-Cas/genética , Línea Celular , Microscopía por Crioelectrón , Edema/patología , Edema/prevención & control , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/uso terapéutico , Semaforinas/química , Semaforinas/genética
2.
Proc Natl Acad Sci U S A ; 117(29): 16938-16948, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32616570

RESUMEN

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.


Asunto(s)
Toxina Diftérica/metabolismo , Endopeptidasas/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Proteolisis , Proteínas de Unión al GTP rap1/metabolismo , Proteínas ras/metabolismo , Animales , Antineoplásicos/uso terapéutico , Células Cultivadas , Toxina Diftérica/química , Toxina Diftérica/genética , Endopeptidasas/química , Endopeptidasas/genética , Femenino , Células HCT116 , Humanos , Masculino , Ratones , Ratones Desnudos , Mutación , Señales de Clasificación de Proteína , Proteínas Recombinantes/uso terapéutico , Proteínas ras/genética
3.
Proc Natl Acad Sci U S A ; 117(11): 5791-5800, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32123106

RESUMEN

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.


Asunto(s)
Antígeno Nuclear de Célula en Proliferación/metabolismo , Ingeniería de Proteínas/métodos , Proteolisis , Ubiquitina-Proteína Ligasas/genética , Sitios de Unión , Células HEK293 , Humanos , Terapia Molecular Dirigida/métodos , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo
4.
Mol Pharm ; 15(11): 5217-5226, 2018 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-30212635

RESUMEN

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery of PNP as well as other cytosolic enzymes implicated in disease.


Asunto(s)
Toxina Diftérica/genética , Sistemas de Liberación de Medicamentos/métodos , Purina-Nucleósido Fosforilasa/administración & dosificación , Purina-Nucleósido Fosforilasa/deficiencia , Proteínas Recombinantes de Fusión/administración & dosificación , Linfocitos B/metabolismo , Citosol/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Enfermedades de Inmunodeficiencia Primaria , Ingeniería de Proteínas , Purina-Nucleósido Fosforilasa/efectos de los fármacos , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/uso terapéutico , Errores Innatos del Metabolismo de la Purina-Pirimidina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T/metabolismo
5.
Proc Natl Acad Sci U S A ; 111(10): 3721-6, 2014 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-24567384

RESUMEN

Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues--clustered between amino acids 1,035 and 1,107--that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.


Asunto(s)
Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Clostridioides difficile/patogenicidad , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Secuencia de Aminoácidos , Toxinas Bacterianas/metabolismo , Clostridioides difficile/genética , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutación/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Estructura Terciaria de Proteína/genética , Radioisótopos de Rubidio/metabolismo
6.
J Biol Chem ; 290(3): 1474-84, 2015 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-25355312

RESUMEN

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.


Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/enzimología , Hidrazonas/química , Malonatos/química , Inhibidores de la Transcriptasa Inversa/química , Antirretrovirales/química , Catálisis , Dominio Catalítico , Evaluación Preclínica de Medicamentos , Iones , Metales/química , Modelos Moleculares , Mutagénesis , Mutación , Unión Proteica , Multimerización de Proteína , Ribonucleasa H/química , Relación Estructura-Actividad
7.
J Biol Chem ; 289(23): 16270-7, 2014 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-24719329

RESUMEN

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor ß-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.


Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Piridinas/farmacología , Pirimidinonas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/antagonistas & inhibidores , Secuencia de Bases , Cinética , Oligodesoxirribonucleótidos
8.
Mol Pharm ; 12(8): 2962-71, 2015 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-26103531

RESUMEN

Platforms enabling targeted delivery of proteins into cells are needed to fully realize the potential of protein-based therapeutics with intracellular sites-of-action. Bacterial toxins are attractive systems to consider as templates for designing protein transduction systems as they naturally bind and enter specific cells with high efficiency. Here we investigated the capacity of diphtheria toxin to function as an intracellular protein delivery vector. We report that diphtheria toxin delivers an impressive array of passenger proteins spanning a range of sizes, structures, and stabilities into cells in a manner that indicates that they are "invisible" to the translocation machinery. Further, we show that α-amylase delivered into cells by a detoxified diphtheria toxin chimera digests intracellular glycogen in live cells, providing evidence that delivered cargo is folded, active, and abundant. The efficiency and versatility of diphtheria toxin over existing systems open numerous possibilities for intracellular delivery of bioactive proteins.


Asunto(s)
Toxina Diftérica/metabolismo , Sistemas de Liberación de Medicamentos , Glucógeno/metabolismo , Fragmentos de Péptidos/metabolismo , alfa-Amilasas/química , alfa-Amilasas/metabolismo , Rastreo Diferencial de Calorimetría , Células HEK293 , Humanos , Pliegue de Proteína
9.
Nat Cancer ; 4(10): 1418-1436, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37697045

RESUMEN

Glioblastoma (GBM) is an incurable brain cancer that lacks effective therapies. Here we show that EAG2 and Kvß2, which are predominantly expressed by GBM cells at the tumor-brain interface, physically interact to form a potassium channel complex due to a GBM-enriched Kvß2 isoform. In GBM cells, EAG2 localizes at neuron-contacting regions in a Kvß2-dependent manner. Genetic knockdown of the EAG2-Kvß2 complex decreases calcium transients of GBM cells, suppresses tumor growth and invasion and extends the survival of tumor-bearing mice. We engineered a designer peptide to disrupt EAG2-Kvß2 interaction, thereby mitigating tumor growth in patient-derived xenograft and syngeneic mouse models across GBM subtypes without overt toxicity. Neurons upregulate chemoresistant genes in GBM cells in an EAG2-Kvß2-dependent manner. The designer peptide targets neuron-associated GBM cells and possesses robust efficacy in treating temozolomide-resistant GBM. Our findings may lead to the next-generation therapeutic agent to benefit patients with GBM.


Asunto(s)
Glioblastoma , Humanos , Ratones , Animales , Glioblastoma/tratamiento farmacológico , Temozolomida/farmacología , Temozolomida/uso terapéutico , Canales de Potasio Éter-A-Go-Go/uso terapéutico , Modelos Animales de Enfermedad , Péptidos/uso terapéutico , Neuronas/patología
10.
J Biol Chem ; 286(34): 29575-83, 2011 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-21737446

RESUMEN

The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre- and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA·DNA primer/template substrates versus DNA·DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA·DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.


Asunto(s)
Cartilla de ADN/química , ADN Viral/química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , VIH-1/enzimología , Indoles/química , Nitrilos/química , Piridonas/química , ARN Viral/química , Sitio Alostérico , Catálisis , Cartilla de ADN/metabolismo , ADN Viral/biosíntesis , Transcriptasa Inversa del VIH/metabolismo , Indoles/metabolismo , Nitrilos/metabolismo , Piridonas/metabolismo , ARN Viral/biosíntesis , Transcripción Reversa/fisiología
11.
J Biol Chem ; 285(35): 26966-26975, 2010 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-20530477

RESUMEN

Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (+)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.


Asunto(s)
Benzoxazinas/química , Farmacorresistencia Viral , Transcriptasa Inversa del VIH/química , VIH-1/enzimología , Mutación Missense , Nevirapina/química , ARN Viral/química , Inhibidores de la Transcriptasa Inversa/química , Ribonucleasa H/química , Alquinos , Sustitución de Aminoácidos , Ciclopropanos , ADN Viral/química , ADN Viral/metabolismo , ADN Viral/farmacología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Nevirapina/farmacología , ARN Viral/biosíntesis , ARN Viral/genética , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/genética , Ribonucleasa H/metabolismo
12.
Sci Adv ; 6(18)2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32917630

RESUMEN

Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-ß1 (ITGB1)-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b (eIF-3b)-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of ITGB1 led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of eIF-3b resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.


Asunto(s)
Toxina Diftérica , Endocitosis , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Endosomas/metabolismo , Humanos , Lisosomas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
13.
Sci Adv ; 6(50)2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33310843

RESUMEN

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.


Asunto(s)
Toxina Diftérica , Terapia de Reemplazo Enzimático , Animales , Encéfalo/metabolismo , Toxina Diftérica/metabolismo , Toxina Diftérica/farmacología , Lisosomas/metabolismo , Ratones , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/metabolismo
14.
Nat Commun ; 9(1): 5233, 2018 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-30531960

RESUMEN

Clostridium difficile is the leading cause of nosocomial diarrhea and colitis in the industrialized world. Disruption of the protective gut microbiota by antibiotics enables colonization by multidrug-resistant C. difficile, which secrete up to three different protein toxins that are responsible for the gastrointestinal sequelae. Oral agents that inhibit the damage induced by toxins, without altering the gut microbiota, are urgently needed to prevent primary disease and break the cycle of antibiotic-induced disease recurrence. Here, we show that the anthelmintic drug, niclosamide, inhibits the pathogenesis of all three toxins by targeting a host process required for entry into colonocytes by each toxin. In mice infected with an epidemic strain of C. difficile, expressing all three toxins, niclosamide reduced both primary disease and recurrence, without disrupting the diversity or composition of the gut microbiota. Given its excellent safety profile, niclosamide may address an important unmet need in preventing C. difficile primary and recurrent diseases.


Asunto(s)
Clostridioides difficile/efectos de los fármacos , Infecciones por Clostridium/prevención & control , Microbioma Gastrointestinal , Niclosamida/farmacología , Animales , Anticestodos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/metabolismo , Células CHO , Células CACO-2 , Línea Celular , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/microbiología , Cricetulus , Células HCT116 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Virulencia/efectos de los fármacos
15.
Biochem Pharmacol ; 142: 13-20, 2017 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-28408344

RESUMEN

Despite enormous efforts, achieving efficacious levels of proteins inside mammalian cells remains one of the greatest challenges in biologics-based drug discovery and development. The inability of proteins to readily cross biological membranes precludes access to the wealth of intracellular targets and applications that lie within mammalian cells. Existing methods of delivery commonly suffer from an inability to target specific cells and tissues, poor endosomal escape, and limited in vivo efficacy. The aim of the present commentary is to highlight the potential of certain classes of bacterial toxins, which naturally deliver a large protein into the cytosolic compartment of target cells after binding a host cell-surface receptor with high affinity, as robust protein delivery platforms. We review the progress made in recent years toward demonstrating the utility of these systems at delivering a wide variety of protein cargo, with special attention paid to three distinct toxin-based platforms. We contend that with recent advances in protein deimmunization strategies, bacterial toxins are poised to introduce biologics into the inner sanctum of cells and treat a wealth of heretofore untreatable diseases with a new generation of therapeutics.


Asunto(s)
Toxinas Bacterianas/química , Portadores de Fármacos/química , Preparaciones Farmacéuticas/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Animales , Toxinas Bacterianas/metabolismo , Citosol/metabolismo , Portadores de Fármacos/metabolismo , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Humanos , Preparaciones Farmacéuticas/metabolismo , Unión Proteica , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/metabolismo
16.
Sci Transl Med ; 8(370): 370tc2, 2016 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-28003550

RESUMEN

New insights into the mechanism of action of ebselen, a small-molecule antivirulence agent that reduces disease pathology in a mouse model of Clostridium difficile infection, suggest a different molecular target may be responsible for its efficacy.


Asunto(s)
Clostridioides difficile , Infecciones por Clostridium , Animales , Antibacterianos , Ratones
17.
Trends Microbiol ; 23(12): 746-748, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26547239

RESUMEN

Effective treatment of Clostridium difficile infections demands a shift away from antibiotics towards toxin-neutralizing agents. Work by Bender et al., using a drug that attenuates toxin action in vivo without affecting bacterial survival, demonstrates the exciting potential of small molecules as a new modality in the fight against C. difficile.


Asunto(s)
Antibacterianos/uso terapéutico , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/tratamiento farmacológico , Virulencia/efectos de los fármacos , Animales
18.
Chem Biol ; 22(2): 175-85, 2015 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-25619932

RESUMEN

Clostridium difficile causes life-threatening diarrhea through the actions of its homologous toxins TcdA and TcdB on human colonocytes. Therapeutic agents that block toxin-induced damage are urgently needed to prevent the harmful consequences of toxin action that are not addressed with current antibiotic-based treatments. Here, we developed an imaging-based phenotypic screen to identify small molecules that protected human cells from TcdB-induced cell rounding. A series of structurally diverse compounds with antitoxin activity were identified and found to act through one of a small subset of mechanisms, including direct binding and sequestration of TcdB, inhibition of endosomal maturation, and noncompetitive inhibition of the toxin glucosyltransferase activity. Distinct classes of inhibitors were used further to dissect the determinants of the toxin-mediated necrosis phenotype occurring at higher doses of toxin. These findings validate and inform novel targeting strategies for discovering small molecule agents to treat C. difficile infection.


Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Clostridioides difficile/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Biflavonoides/química , Biflavonoides/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Colatos/química , Colatos/metabolismo , Ácido Gálico/análogos & derivados , Ácido Gálico/química , Ácido Gálico/metabolismo , Humanos , Cinética , Necrosis , Floretina/química , Floretina/metabolismo , Unión Proteica , Bibliotecas de Moléculas Pequeñas/metabolismo , Células Vero
19.
Antivir Ther ; 16(7): 1123-30, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22024528

RESUMEN

BACKGROUND: HIV-HBV-coinfected individuals who need to be treated only for their HBV infection have limited therapeutic options, since most approved anti-HBV agents have a risk of selecting for drug-resistant HIV mutants. In vivo data are inconclusive as to whether telbivudine (LdT) may exert antiviral effects against HIV. Thus, we investigated in further detail the antiviral activity and the biochemical properties of LdT against HIV-1. METHODS: To investigate the activity of LdT against HIV-1 in humans we analysed viral dynamics and genotypic and phenotypic resistance development in two HIV-HBV-coinfected individuals with no prior antiviral exposure. To investigate the activity of LdT against HIV-1 in vitro, LdT susceptibility for HIV-1 wild-type strains as well as drug-resistant strains was determined. Furthermore, we studied whether the 5'-triphosphate form of LdT (LdT-TP) can act as a substrate for wild-type HIV-1 RT. RESULTS: In the two patients studied, LdT treatment did not result in a significant decline of HIV-1 RNA load nor in selection of genotypic or phenotypic resistance in HIV-1 RT. In vitro virological analyses demonstrated that LdT had no activity (50% effective concentration >100 µM) against wild type HIV and drug-resistant variants. Biochemical analyses demonstrated that LdT-TP is not incorporated by wild-type HIV-1 RT. CONCLUSIONS: Based on the in vivo and in vitro evidence obtained in this study, we conclude that LdT has no anti-HIV-1 activity and is currently the only selective anti-HBV agent among the five FDA-approved nucleoside/nucleotide analogues for treatment of HBV infections in HIV-infected individuals.


Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Nucleósidos/farmacología , Pirimidinonas/farmacología , Adulto , Antivirales/uso terapéutico , Recuento de Linfocito CD4 , Línea Celular , Coinfección , ADN Viral/sangre , Genotipo , Células HEK293 , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , VIH-1/fisiología , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/virología , Humanos , Masculino , Persona de Mediana Edad , Nucleósidos/sangre , Nucleósidos/uso terapéutico , Fenotipo , Pirimidinonas/sangre , Pirimidinonas/uso terapéutico , ARN Viral/sangre , Telbivudina , Timidina/análogos & derivados , Carga Viral
20.
Viruses ; 2(4): 900-926, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21994660

RESUMEN

Since the human immunodeficiency virus (HIV) was discovered as the etiological agent of acquired immunodeficiency syndrome (AIDS), it has encouraged much research into antiviral compounds. The reverse transcriptase (RT) of HIV has been a main target for antiviral drugs. However, all drugs developed so far inhibit the polymerase function of the enzyme, while none of the approved antiviral agents inhibit specifically the necessary ribonuclease H (RNase H) function of RT. This review provides a background on structure-function relationships of HIV-1 RNase H, as well as an outline of current attempts to develop novel, potent chemotherapeutics against a difficult drug target.

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