RESUMEN
CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Modelos Biológicos , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética/genética , Empalme Alternativo/genética , Supervivencia Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Activación Enzimática/genética , Células HL-60 , Humanos , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The assembly of lipopolysaccharide (LPS) on the surface of Gram-negative bacterial cells is essential for their viability and is achieved by the seven-protein LPS transport (Lpt) pathway. The outer membrane (OM) lipoprotein LptE and the ß-barrel membrane protein LptD form a complex that assembles LPS into the outer leaflet of the OM. We report a crystal structure of the Escherichia coli OM lipoprotein LptE at 2.34 Å. The structure reveals homology to eukaryotic LPS-binding proteins and allowed for the prediction of an LPS-binding site, which was confirmed by genetic and biophysical experiments. Specific point mutations at this site lead to defects in OM biogenesis. We show that wild-type LptE disrupts LPS-LPS interactions in vitro and that these mutations decrease the ability of LptE to disaggregate LPS. Transmission electron microscopic imaging shows that LptE can disrupt LPS aggregates even at substoichiometric concentrations. We propose a model in which LptE functions as an LPS transfer protein in the OM translocon by disaggregating LPS during transport to allow for its insertion into the OM.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipopolisacáridos/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión/genética , Transporte Biológico/fisiología , Catálisis , Cristalización , Proteínas de Escherichia coli/genética , Microscopía Electrónica de Transmisión , Complejos Multiproteicos/genéticaRESUMEN
Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.
Asunto(s)
Arilsulfotransferasa/química , Proteínas de Escherichia coli/química , Oxidorreductasas/química , Proteína Disulfuro Isomerasas/química , Secuencia de Aminoácidos , Arilsulfotransferasa/fisiología , Catálisis , Cristalografía por Rayos X , Disulfuros/química , Proteínas de Escherichia coli/fisiología , Humanos , Datos de Secuencia Molecular , Oxidorreductasas/fisiología , Proteína Disulfuro Isomerasas/fisiología , Pliegue de Proteína , Especificidad por Sustrato , Difracción de Rayos X/métodosRESUMEN
Making your (Dsb) connection: the redox pathway bringing reducing equivalents from bacterial cytoplasm, across the inner membrane, to the three reductive Dsb pathways in the otherwise oxidizing periplasm (see scheme; TR=thioredoxin reductase, Trx=thioredoxin) is reconstituted from purified components. Transfer of reducing equivalents across the membrane is demonstrated and underlying mechanistic details are revealed.
Asunto(s)
Membrana Celular/metabolismo , Citoplasma/metabolismo , Disulfuros/química , Escherichia coli/metabolismo , Periplasma/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo , Oxidación-Reducción , Proteolípidos/metabolismo , Transducción de SeñalRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic human disease, but to date, only one therapy (tolvaptan) is approved to treat kidney cysts in ADPKD patients. Cyclin-dependent kinase 5 (CDK5), an atypical member of the cyclin-dependent kinase family, has been implicated as a target for treating ADPKD. However, no compounds have been disclosed to date that selectively inhibit CDK5 while sparing the broader CDK family members. Herein, we report the discovery of CDK5 inhibitors, including GFB-12811, that are highly selective over the other tested kinases. In cellular assays, our compounds demonstrate CDK5 target engagement while avoiding anti-proliferative effects associated with inhibiting other CDKs. In addition, we show that the compounds in this series exhibit promising in vivo PK profiles, enabling their use as tool compounds for interrogating the role of CDK5 in ADPKD and other diseases.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Diseño de Fármacos , Descubrimiento de Drogas , Células HEK293 , Humanos , Modelos Moleculares , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 (ClinicalTrials.gov Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.
Asunto(s)
Neoplasias de la Mama , Ciclo Celular , Quinasas Ciclina-Dependientes , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas , Animales , Femenino , Humanos , Ratones , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pharmacodynamic (PD) studies are an essential component of preclinical drug discovery. Current approaches for PD studies, including the analysis of novel kidney disease targeting therapeutic agents, are limited to animal models with unclear translatability to the human condition. To address this challenge, we developed a novel approach for PD studies using transplanted, perfused human kidney organoids. We performed pharmacokinetic (PK) studies with GFB-887, an investigational new drug now in phase 2 trials. Orally dosed GFB-887 to athymic rats that had undergone organoid transplantation resulted in measurable drug exposure in transplanted organoids. We established the efficacy of orally dosed GFB-887 in PD studies, where quantitative analysis showed significant protection of kidney filter cells in human organoids and endogenous rat host kidneys. This widely applicable approach demonstrates feasibility of using transplanted human organoids in preclinical PD studies with an investigational new drug, empowering organoids to revolutionize drug discovery.
Asunto(s)
Enfermedades Renales , Organoides , Animales , Descubrimiento de Drogas , Drogas en Investigación , Humanos , Riñón , RatasRESUMEN
Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3'-phosphate-5'-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a critical step in hepatic detoxification and extracellular signaling. In contrast, little is known about sulfotransferases in bacteria, which make use of sulfurylated molecules as mediators of cell-cell interactions and host-pathogen interactions. Bacterial arylsulfate sulfotransferases (also termed aryl sulfotransferases), in contrast to PAPS-dependent sulfotransferases, transfer sulfuryl groups exclusively among phenolic compounds in a PAPS-independent manner. Here, we report the crystal structure of the virulence factor arylsulfate sulfotransferase (ASST) from the prototypic, pyelonephritogenic Escherichia coli strain CFT073 at 2.0-A resolution, and 2 catalytic intermediates, at 2.1-A and 2.4-A resolution, with substrates bound in the active site. ASST is one of the largest periplasmic enzymes and its 3D structure differs fundamentally from all other structurally characterized sulfotransferases. Each 63.8-kDa subunit of the ASST homodimer comprises a 6-bladed beta-propeller domain and a C-terminal beta-sandwich domain. The active sites of the dimer are situated at the center of the channel formed by each beta-propeller and are defined by the side chains of His-252, His-356, Arg-374, and His-436. We show that ASST follows a ping-pong bi-bi reaction mechanism, in which the catalytic residue His-436 undergoes transient sulfurylation, a previously unreported covalent protein modification. The data provide a framework for understanding PAPS-independent sulfotransfer and a basis for drug design targeting this bacterial virulence factor.
Asunto(s)
Arilsulfotransferasa/química , Arilsulfotransferasa/metabolismo , Escherichia coli/enzimología , Animales , Arilsulfotransferasa/genética , Dominio Catalítico , Cristalografía , Dimerización , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Cinética , Mamíferos , Mutagénesis Sitio-Dirigida , Fosfoadenosina Fosfosulfato/metabolismo , Estructura Terciaria de Proteína , Pielonefritis/microbiología , Relación Estructura-Actividad , Especificidad por Sustrato , Factores de Virulencia/química , Factores de Virulencia/metabolismoRESUMEN
Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.
Asunto(s)
Calmodulina/metabolismo , Moduladores del Transporte de Membrana/farmacología , Piridazinas/farmacología , Canales Catiónicos TRPC/efectos de los fármacos , Proteínas de Pez Cebra/efectos de los fármacos , Animales , Sitios de Unión , Calmodulina/química , Calmodulina/genética , Células HEK293 , Humanos , Ligandos , Potenciales de la Membrana , Moduladores del Transporte de Membrana/química , Moduladores del Transporte de Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Piridazinas/química , Piridazinas/metabolismo , Células Sf9 , Relación Estructura-Actividad , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Xenopus , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The nonselective Ca2+-permeable transient receptor potential (TRP) channels play important roles in diverse cellular processes, including actin remodeling and cell migration. TRP channel subfamily C, member 5 (TRPC5) helps regulate a tight balance of cytoskeletal dynamics in podocytes and is suggested to be involved in the pathogenesis of proteinuric kidney diseases, such as focal segmental glomerulosclerosis (FSGS). As such, protection of podocytes by inhibition of TRPC5 mediated Ca2+ signaling may provide a novel therapeutic approach for the treatment of proteinuric kidney diseases. Herein, we describe the identification of a novel TRPC5 inhibitor, GFB-8438, by systematic optimization of a high-throughput screening hit, pyridazinone 1. GFB-8438 protects mouse podocytes from injury induced by protamine sulfate (PS) in vitro. It is also efficacious in a hypertensive deoxycorticosterone acetate (DOCA)-salt rat model of FSGS, significantly reducing both total protein and albumin concentrations in urine.
RESUMEN
Disulfide bond formation is a critical step in the folding of many secretory proteins. In bacteria, disulfide bonds are introduced by the periplasmic dithiol oxidase DsbA, which transfers its catalytic disulfide bond to folding polypeptides. Reduced DsbA is reoxidized by ubiquinone Q8, catalyzed by inner membrane quinone reductase DsbB. Here, we report the preparation of a kinetically stable ternary complex between wild-type DsbB, containing all essential cysteines, Q8 and DsbA covalently bound to DsbB. The crystal structure of this trapped DsbB reaction intermediate exhibits a charge-transfer interaction between Q8 and the Cys44 in the DsbB reaction center providing experimental evidence for the mechanism of de novo disulfide bond generation in DsbB.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de la Membrana/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Membrana Celular/metabolismo , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Disulfuros/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Oxidación-Reducción , Estructura Secundaria de ProteínaRESUMEN
The outer membrane (OM) in Gram-negative bacteria is an asymmetric bilayer with mostly lipopolysaccharide (LPS) molecules in the outer leaflet. During OM biogenesis, new LPS molecules are transported from their site of assembly on the inner membrane to the OM by seven LPS transport proteins (LptA-G). The complex formed between the integral ß-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasmic side of the OM to its final location on the cell surface. Because of its essential function in many Gram-negative bacteria, the LPS transport pathway is an interesting target for the development of new antibiotics. A family of macrocyclic peptidomimetics was discovered recently that target LptD and inhibit LPS transport specifically in Pseudomonas spp. The related molecule Murepavadin is in clinical development for the treatment of life-threatening infections caused by P. aeruginosa. To characterize the interaction of these antibiotics with LptD from P. aeruginosa, we characterized the binding site by cross-linking to a photolabeling probe. We used a hypothesis-free mass spectrometry-based proteomic approach to provide evidence that the antibiotic cross-links to the periplasmic segment of LptD, containing a ß-jellyroll domain and an N-terminal insert domain characteristic of Pseudomonas spp. Binding of the antibiotic to the periplasmic segment is expected to block LPS transport, consistent with the proposed mode of action and observed specificity of these antibiotics. These insights may prove valuable for the discovery of new antibiotics targeting the LPS transport pathway in other Gram-negative bacteria.
Asunto(s)
Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Peptidomiméticos/metabolismo , Pseudomonas aeruginosa/química , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Bacterias Gramnegativas/efectos de los fármacos , Lipopolisacáridos/metabolismo , Periplasma , Dominios Proteicos , Transporte de ProteínasRESUMEN
The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG precursor pool but does not weaken cell wall integrity, challenging models of precursor sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface without impairing OM function. Because LPS* contains the canonical vancomycin binding site, these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns (PAMPs).
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Ligasas de Carbono-Oxígeno/genética , Pared Celular/química , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Lipopolisacáridos/metabolismo , Peptidoglicano/metabolismo , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión , Ligasas de Carbono-Oxígeno/química , Ligasas de Carbono-Oxígeno/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glicosilación , Lipopolisacáridos/química , Mutación , Peptidoglicano/química , Vancomicina/farmacología , Resistencia a la Vancomicina/genéticaRESUMEN
N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is essential in eukaryotes and catalyzed by oligosaccharyl transferase (OST). Human OST is a hetero-oligomer of seven subunits. The subunit N33/Tusc3 is a tumor suppressor candidate, and defects in the subunit N33/Tusc3 are linked with nonsyndromic mental retardation. Here, we show that N33/Tusc3 possesses a membrane-anchored N-terminal thioredoxin domain located in the ER lumen that may form transient mixed disulfide complexes with OST substrates. X-ray structures of complexes between N33/Tusc3 and two different peptides as model substrates reveal a defined peptide-binding groove adjacent to the active site that can accommodate peptides in opposite orientations. Structural and biochemical data show that N33/Tusc3 prefers peptides bearing a hydrophobic residue two residues away from the cysteine forming the mixed disulfide with N33/Tusc3. Our results support a model in which N33/Tusc3 increases glycosylation efficiency for a subset of human glycoproteins by slowing glycoprotein folding.
Asunto(s)
Disulfuros/química , Proteínas de la Membrana/química , Péptidos/química , Subunidades de Proteína/química , Proteínas Supresoras de Tumor/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Expresión Génica , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Proteínas Supresoras de Tumor/genéticaRESUMEN
Sulfurylation of biomolecules (often termed sulfonation or sulfation) has been described in many organisms in all kingdoms of life. To date, most studies on sulfotransferases, the enzymes catalyzing sulfurylation, have focused on 3'-phosphate-5'-phosphosulfate (PAPS)-dependent enzymes, which transfer the sulfuryl group from this activated anhydride to hydroxyl groups of acceptor molecules. By contrast, the PAPS-independent aryl sulfotransferases (ASSTs) from bacteria, which catalyze sulfotransfer from phenolic sulfate esters to another phenol in the bacterial periplasm, were not well characterized until recently, although they were first described in 1986 in a search for nonhepatic sulfurylation processes. Recent studies revealed that this unusual class of sulfotransferases differs profoundly in both molecular structure and catalytic mechanism from PAPS-dependent sulfotransferases, and that ASSTs from certain bacterial pathogens are upregulated during infection. In this review, we summarize the literature on the roles of sulfurylation in prokaryotes and analyze the occurrence of ASSTs and their dependence on Dsb proteins catalyzing oxidative folding in the periplasm. Furthermore, we discuss structural differences and similarities between aryl sulfotransferases and PAPS-dependent sulfotransferases.
Asunto(s)
Arilsulfotransferasa/metabolismo , Bacterias/metabolismo , Bacterias/patogenicidad , Disulfuros/metabolismo , Fosfoadenosina Fosfosulfato/metabolismo , Bacterias/enzimología , Disulfuros/química , Humanos , Oxidación-Reducción , Pliegue de ProteínaRESUMEN
Disulfide bond formation in the Escherichia coli periplasm requires the transfer of electrons from substrate proteins to DsbA, which is recycled as an oxidant by the membrane protein DsbB. The highly virulent, uropathogenic E. coli strain CFT073 contains a second, homologous pair of proteins, DsbL and DsbI, which are encoded in a tri-cistronic operon together with a periplasmic, uropathogen-specific arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI form a functional redox pair, and that ASST is a substrate of DsbL/DsbI in vivo. DsbL is the most reactive oxidizing thioredoxin-like protein known to date. In contrast to DsbA, however, DsbL oxidizes reduced RNaseA with a much lower rate and prevents unspecific aggregation of reduced insulin. The 1.55 A resolution crystal structure of reduced DsbL provides insight into the reduced state of thioredoxin-like dithiol oxidases at high resolution, and reveals an unusual cluster of basic residues stabilizing the thiolate anion of the nucleophilic active-site cysteine. We propose that the DsbL/DsbI pair of uropathogenic E. coli was acquired as an additional, specific redox couple that guarantees biological activity of ASST.
Asunto(s)
Arilsulfotransferasa/metabolismo , Disulfuros/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Oxidorreductasas/metabolismo , Periplasma/enzimología , Secuencia de Aminoácidos , Arilsulfotransferasa/química , Arilsulfotransferasa/genética , Sitios de Unión , Cristalografía por Rayos X , Disulfuros/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Prueba de Complementación Genética , Glutatión/metabolismo , Enlace de Hidrógeno , Insulina/química , Insulina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Conformación Proteica , Estructura Terciaria de Proteína , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/metabolismo , Alineación de SecuenciaRESUMEN
The new bis-phenanthridine triamine is characterised by three pK(a) values: 3.65; 6.0 and >7.5. A significant difference in the protonation state of at pH = 5 (four positive charges) and at pH = 7 (less than two positive charges) accounts for the strong dependence of -nucleotide binding constants on nucleotide charge under acidic conditions, whereas at neutral pH all -nucleotide complexes are of comparable stability. All experimental data point at intercalation as the dominant binding mode of to polynucleotides. However, there is no indication of bis-intercalation of the two phenanthridine subunits in binding to double stranded polynucleotides, the respective complexes being most likely mono-intercalative. Thermal stabilisation of calf thymus DNA (ct-DNA) and poly A-poly U duplexes upon addition of is significantly higher at pH = 5 than at neutral conditions. This is not the case with poly dA-poly dT, indicating that the specific secondary structure of the latter, most likely the shape of the minor groove, plays a key role in complex stability. At pH = 5 acts as a fluorimetric probe for poly G (emission quenching) as opposed to other ss-polynucleotides (emission increase), while at neutral conditions this specificity is lost. One order of magnitude higher cytotoxicity of compared to its "monomer" can be accounted for by cooperative action of two phenanthridinium units and the charged triamine linker. The results presented here are of interest to the development of e.g. sequence-selective cytostatic drugs, and in particular for the possibility to control the drug activity properties over binding to DNA and/or RNA by variation of the pH of its surrounding.