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1.
Nature ; 602(7897): 529-533, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35140402

RESUMO

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, ß-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αß, α4ßδ, α6ßδ and α5ßγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1ßγ, α2ßγ and α3ßγ receptor responses5,7-12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αß GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA-Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αß receptors that adapt them to a role in tonic signalling.


Assuntos
Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Receptores de GABA-A , Animais , Proteínas Neurotóxicas de Elapídeos , Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Mamíferos/metabolismo , Inibição Neural/fisiologia , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Zinco , Ácido gama-Aminobutírico/metabolismo
3.
Nature ; 535(7613): 517-522, 2016 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-27437577

RESUMO

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.


Assuntos
Espaço Extracelular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Anilidas/química , Anilidas/metabolismo , Anilidas/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação/genética , Colesterol/metabolismo , Colesterol/farmacologia , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica/genética , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened
4.
Nature ; 512(7514): 270-5, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-24909990

RESUMO

Type-A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human ß3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission.


Assuntos
Receptores de GABA-A/química , Benzamidinas/química , Benzamidinas/metabolismo , Benzamidinas/farmacologia , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Desenho de Fármacos , Agonistas de Receptores de GABA-A/química , Agonistas de Receptores de GABA-A/metabolismo , Agonistas de Receptores de GABA-A/farmacologia , Predisposição Genética para Doença , Glicosilação , Humanos , Modelos Moleculares , Mutação/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Receptores de GABA-A/genética , Transmissão Sináptica
5.
Biochemistry ; 54(13): 2270-82, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25768916

RESUMO

Platinum-derivatized homopyrimidine triplex-forming oligonucleotides (Pt-TFOs) consisting of 2'-O-methyl-5-methyluridine, 2'-O-methyl-5-methylcytidine, and a single 3'-N7-trans-chlorodiammine platinum(II)-2'-deoxyguanosine were designed to cross-link to the transcribed strand at four different sequences in the human androgen receptor (AR) gene. Fluorescence microscopy showed that a fluorescein-tagged Pt-TFO localizes in both the cytoplasm and nucleus when it is transfected into LAPC-4 cells, a human prostate cancer cell line, using Lipofectamine 2000. A capture assay employing streptavidin-coated magnetic beads followed by polymerase chain reaction (PCR) amplification was used to demonstrate that 5'-biotin-conjugated Pt-TFOs cross-link in vitro to their four designated AR gene targets in genomic DNA extracted from LAPC-4 cells. Similarly, the capture assay was used to examine cross-linking between the 5'-biotin-conjugated Pt-TFOs and the AR gene in LAPC-4 cells in culture. Three of the four Pt-TFOs cross-linked to their designated target, suggesting that different regions of the AR gene are not uniformly accessible to Pt-TFO cross-linking. LAPC-4 cells were transfected with fluorescein-tagged Pt-TFO or a control oligonucleotide that does not bind or cross-link to AR DNA. The levels of AR mRNA in highly fluorescent cells isolated by fluorescence-activated cell sorting were determined by RT-qPCR, and the levels of AR protein were monitored by immunofluorescence microscopy. Decreases in mRNA and protein levels of 40 and 30%, respectively, were observed for fluorescein-tagged Pt-TFO versus control treated cells. Although the levels of knockdown of AR mRNA and protein were modest, the results suggest that Pt-TFOs hold potential as agents for controlling gene expression by cross-linking to DNA and disrupting transcription.


Assuntos
Técnicas de Silenciamento de Genes , Oligonucleotídeos/química , Receptores Androgênicos/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Fluoresceína/química , Corantes Fluorescentes/química , Glutationa/química , Humanos , Masculino , Microscopia de Fluorescência , Terapia de Alvo Molecular/métodos , Oligonucleotídeos/farmacologia , Compostos Organoplatínicos/química , Reação em Cadeia da Polimerase/métodos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Transfecção/métodos
6.
Lancet Oncol ; 15(1): 96-105, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24332516

RESUMO

BACKGROUND: Basal-cell carcinoma is the most common form of skin cancer and its incidence is increasing worldwide. We aimed to assess the effectiveness of imiquimod cream versus surgical excision in patients with low-risk basal-cell carcinoma. METHODS: We did a multicentre, parallel-group, pragmatic, non-inferiority, randomised controlled trial at 12 centres in the UK, in which patients were recruited between June 19, 2003, and Feb 22, 2007, with 3 year follow-up from June 26, 2006, to May 26, 2010. Participants of any age were eligible if they had histologically confirmed primary nodular or superficial basal-cell carcinoma at low-risk sites. We excluded patients with morphoeic or recurrent basal-cell carcinoma and those with Gorlin syndrome. Participants were randomly assigned (1:1) via computer-generated blocked randomisation, stratified by centre and tumour type, to receive either imiquimod 5% cream once daily for 6 weeks (superficial) or 12 weeks (nodular), or surgical excision with a 4 mm margin. The randomisation sequence was concealed from study investigators. Because of the nature of the interventions, masking of participants was not possible and masking of outcome assessors was only partly possible. The trial statistician was masked to allocation until all analyses had been done. The primary outcome was the proportion of participants with clinical success, defined as absence of initial treatment failure or signs of recurrence at 3 years from start of treatment. We used a prespecified non-inferiority margin of a relative risk (RR) of 0.87. Analysis was by a modified intention-to-treat population and per protocol. This study is registered as an International Standard Randomised Controlled Trial (ISRCTN48755084), and with ClinicalTrials.gov, number NCT00066872. FINDINGS: 501 participants were randomly assigned to the imiquimod group (n=254) or the surgical excision group (n=247). At year 3, 401 (80%) patients were included in the modified intention-to-treat group. At 3 years, 178 (84%) of 213 participants in the imiquimod group were treated successfully compared with 185 (98%) of 188 participants in the surgery group (RR 0.84, 98% CI 0.78-0.91; p<0.0001). No clear difference was noted between groups in patient-assessed cosmetic outcomes. The most common adverse events were itching (211 patients in the imiquimod group vs 129 in the surgery group) and weeping (160 vs 81). We recorded serious adverse events in 99 (40%) of 249 participants in the imiquimod group and 97 (42%) of 229 in the surgery group had serious adverse events, but none were regarded as related to treatment. 12 (5%) participants in the imiquimod group withdrew because of adverse events compared with four (2%) in the surgery group. INTERPRETATION: Imiquimod was inferior to surgery according to our predefined non-inferiority criterion. Although excisional surgery remains the best treatment for low-risk basal-cell carcinoma, imiquimod cream might still be a useful treatment option for small low-risk superficial or nodular basal-cell carcinoma dependent on factors such as patient preference, size and site of the lesion, and whether the patient has more than one lesion. FUNDING: Cancer Research UK.


Assuntos
Aminoquinolinas/administração & dosagem , Antineoplásicos/administração & dosagem , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/cirurgia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/cirurgia , Idoso , Aminoquinolinas/efeitos adversos , Feminino , Humanos , Imiquimode , Masculino , Pessoa de Meia-Idade , Pomadas
8.
Nat Chem Biol ; 7(3): 182-8, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21278739

RESUMO

Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.


Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diterpenos/farmacologia , Fenantrenos/farmacologia , Fator de Transcrição TFIIH/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , DNA Helicases/química , Proteínas de Ligação a DNA/química , Diterpenos/química , Diterpenos/isolamento & purificação , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Compostos de Epóxi/química , Compostos de Epóxi/isolamento & purificação , Compostos de Epóxi/farmacologia , Células HeLa , Humanos , Fenantrenos/química , Fenantrenos/isolamento & purificação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/química
9.
Nat Struct Mol Biol ; 30(12): 1936-1946, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37903907

RESUMO

α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the ß-carboline, methyl 6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an 'upper' and 'lower' moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure-activity relationships, and empower future structure-based drug design campaigns.


Assuntos
Receptores de GABA-A , Ácido gama-Aminobutírico , Receptores de GABA-A/metabolismo , Isoxazóis/farmacologia
10.
J Biol Inorg Chem ; 17(8): 1197-208, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22965663

RESUMO

Platinated triplex-forming oligonucleotides (TFOs) consisting of 2'-methoxythymidine and 2'-methoxy-5-methylcytidine and an N-7 platinated deoxyguanosine ((Pt)G) at the 5'-((Pt)G-TFO), 3'-(TFO-G(Pt)), or 3'- and 5'-((Pt)G-TFO-G(Pt)) ends of the TFO form mono-((Pt)G-TFO and TFO-G(Pt)) and interstrand ((Pt)G-TFO-G(Pt)) cross-links with target DNA as a result of reaction of the (Pt)G with guanines adjacent to the homopurine TFO binding site in the target. The extent of cross-linking is greatest when the (Pt)G is located on the 3' end of the TFO and the target guanine is on the same strand as the TFO binding site. Multiple, contiguous deoxyguanosines in the TFO binding site or a cytosine adjacent to the G(Pt) of the TFO significantly reduce cross-linking. DNA reporter plasmids in which platinated TFOs were cross-linked at a site in the transcribed region between a CMV promoter and a luciferase reporter gene were transfected into Chinese hamster ovary cells, and luciferase expression was compared with that for the corresponding non-cross-linked plasmid. Luciferase expression was inhibited 95 % when TFO-G(Pt) was bound and cross-linked to the transcribed strand, demonstrating that the cross-linked TFO was able to block transcription elongation. Further inhibition (99 %) was observed in nucleotide excision repair (NER) deficient cells, suggesting that NER may repair this lesion. The 3'-G(Pt) group of TFO-G(Pt) protects the TFO from degradation by exonucleases found in mammalian serum. Taken together, these results suggest that platinated TFOs of the type TFO-G(Pt) may find applications as agents for suppressing DNA transcription and consequently inhibiting gene expression in mammalian cells.


Assuntos
DNA/química , Oligonucleotídeos/farmacologia , Platina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Células CHO , Cricetinae , Reagentes de Ligações Cruzadas/farmacologia , DNA/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Estabilidade de Medicamentos , Guanina/química , Dados de Sequência Molecular , Estrutura Molecular , Temperatura
11.
Biochemistry ; 50(5): 882-90, 2011 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-21174443

RESUMO

Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.


Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Alquilantes/química , Pareamento de Bases , Sequência de Bases , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico
12.
Biochemistry ; 49(18): 3977-88, 2010 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-20373772

RESUMO

DNA interstrand cross-links (ICLs) are cytotoxic products of common anticancer drugs and cellular metabolic processes, whose mechanism(s) of repair remains poorly understood. In this study, we show that cross-link structure affects ICL repair in nonreplicating reporter plasmids that contain a mispaired N(4)C-ethyl-N(4)C (C-C), N3T-ethyl-N3T (T-T), or N1I-ethyl-N3T (I-T) ICL. The T-T and I-T cross-links obstruct the hydrogen bond face of the base and mimic the N1G-ethyl-N3C ICL created by bis-chloroethylnitrosourea, whereas the C-C cross-link does not interfere with base pair formation. Host-cell reactivation (HCR) assays in human and hamster cells showed that repair of these ICLs primarily involves the transcription-coupled nucleotide excision repair (TC-NER) pathway. Repair of the C-C ICL was 5-fold more efficient than repair of the T-T or I-T ICLs, suggesting the latter cross-links hinder lesion bypass following initial ICL unhooking. The level of luciferase expression from plasmids containing a C-C cross-link remnant on either the transcribed or nontranscribed strand increased in NER-deficient cells, indicating NER involvement occurs at a step prior to remnant removal, whereas expression from similar T-T remnant plasmids was inhibited in NER-deficient cells, demonstrating NER is required for remnant removal. Sequence analysis of repaired plasmids showed a high proportion of C residues inserted at the site of the T-T and I-T cross-links, and HCR assays showed that Rev1 was likely responsible for these insertions. In contrast, both C and G residues were inserted at the C-C cross-link site, and Rev1 was not required for repair, suggesting replicative or other translesion polymerases can bypass the C-C remnant.


Assuntos
Reparo do DNA , Replicação do DNA , DNA/química , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Cricetinae , Ciclofosfamida , DNA/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Doxorrubicina , Células HeLa , Humanos , Conformação de Ácido Nucleico , Recombinação Genética , Vincristina
13.
Bioconjug Chem ; 20(12): 2222-30, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19950917

RESUMO

Triplex-forming oligonucleotides (TFOs) can bind to polypurine x polypyrimidine tracts in DNA and, as a consequence, perturb the normal functioning of a targeted gene. The effectiveness of such antigene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here, we report that attachment of N-7-platinated guanine nucleosides to the 3'- and/or 5'-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3',5'-bis-platinated TFOs can result in the formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37 degrees C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast, high levels (approximately 86%) of adduct formation by platinated 2'-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2'-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinum-conjugated TFOs may be good candidates for use as antigene agents.


Assuntos
Cisplatino/química , DNA/química , Oligonucleotídeos/química , Compostos Organometálicos/química , Concentração de Íons de Hidrogênio , Cinética , Oligonucleotídeos/síntese química , Compostos Organometálicos/síntese química , Platina/química
14.
J Biol Inorg Chem ; 14(6): 873-81, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19350290

RESUMO

Triplex-forming oligonucleotides (TFOs) can bind specifically to polypurine sequences in double-stranded DNA. A single interruption of this polypurine tract can greatly destabilize triplex formation. The stability of triplexes can be significantly enhanced by covalently linking the TFO to its DNA target with reactive functional groups conjugated to the TFO. Covalently cross-linked TFOs are effective inhibitors of transcription of the target DNA sequence. We have designed a TFO with a platinum-modified base that can interact with and cross-link to a cytosine interruption in the polypurine tract of a target DNA duplex. The TFO contains an N(4)-(aminoalkyl)cytosine derivatized with cis-diamminediaquaplatinum(II) or trans-diamminediaquaplatinum(II). When bound to its target, the tethered platinum of the TFO can reach across the major groove and form an adduct with the guanine N7 of the interrupting C.G base pair. The optimal tether length is five methylene groups, and cross-linking is most efficient when the tether is modified with trans-diamminediaquaplatinum(II). Cross-linking requires that the TFO is bound to its designated DNA target. Addition of cyanide to the cross-linked TFO product reversed the cross-link, behavior that is consistent with the presence of a platinum-guanine adduct. The kinetics of the cross-linking reaction were studied and the half-life of the cross-linking reaction was approximately 3 h. Our results demonstrate that platinum-conjugated TFOs can be designed to cross-link with DNA targets that contain a single pyrimidine interruption. Modifications of this type may prove useful for expanding the DNA sequences that can be targeted by TFOs and increasing the stability of the resulting triplexes.


Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , DNA/genética , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Platina/química , Nucleotídeos de Purina/química , Sequência de Bases , Cianetos/química , Citosina , DNA/metabolismo , Cinética , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Nucleotídeos de Purina/genética , Especificidade por Substrato , Temperatura de Transição
15.
Chem Res Toxicol ; 22(7): 1285-97, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19580249

RESUMO

DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.


Assuntos
Reagentes de Ligações Cruzadas/química , Reparo do DNA , DNA/química , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Células HeLa , Humanos , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/metabolismo , Análise de Sequência de DNA
16.
Nucleic Acids Res ; 35(22): e152, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18073193

RESUMO

The repair of DNA by nucleotide excision repair (NER) and non-homologous end joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Examination of NHEJ and NER in vitro using cell-free extracts has led to a deeper understanding of the biochemical mechanisms that underlie these processes. Current methods for production of whole-cell extracts (WCEs) to investigate NER and NHEJ start with one or more liters of culture containing 1-5 x 10(9) cells. Here, we describe a small-scale method for production of WCE that can be used to study NER. We also describe a rapid, small-scale method for the preparation of WCE that can be used in the study of NHEJ. These methods require less time, 20- to 1000-fold fewer cells than large-scale extracts, facilitate examination of numerous samples and are ideal for such applications as the study of host-virus interactions and analysis of mutant cell lines.


Assuntos
Extratos Celulares/genética , Reparo do DNA , Adenovírus Humanos/fisiologia , Animais , Células CHO , Técnicas de Cultura de Células , Linhagem Celular , Sistema Livre de Células , Cricetinae , Cricetulus , Células HeLa , Humanos , Recombinação Genética
17.
Biochemistry ; 47(48): 12931-8, 2008 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-18991396

RESUMO

The cancer chemotherapeutic agent cis-diamminedichloroplatinum(II) or cisplatin reacts primarily with guanines in DNA to form 1,2-Pt-GG and 1,3-Pt-GNG intrastrand cross-links and, to a lesser extent, G-G interstrand cross-links. Recent NMR evidence has suggested that cisplatin can also form a coordination complex with the phosphodiester internucleotide linkage of DNA. We have examined the effects of the phosphodiester backbone on the reactions of cisplatin with oligodeoxyribonucleotides that lack or contain a GTG sequence. Cisplatin forms a stable adduct with TpT that can be isolated by reversed phase HPLC. The cis-Pt-TpT adduct contains a single Pt, as determined by atomic absorption spectroscopy (AAS) and by electrospray ionization mass spectrometry (ESI-MS), and is resistant to digestion by snake venom phosphodiesterase. Treatment of the adduct with sodium cyanide regenerates TpT. Similar adduct formation was observed when T(pT)(8) was treated with cisplatin, but not when the phosphodiester linkages of T(pT)(8) were replaced with methylphosphonate groups. These results suggest that the platinum may be coordinated with the oxygens of the thymine and possibly with those of the phosphodiester group. As expected, reaction of a 9-mer containing a GTG sequence with cisplatin yielded an adduct that contained a 1,3-Pt-GTG intrastrand cross-link. However, we found that the number and placement of phosphodiesters surrounding a GTG sequence significantly affected intrastrand cross-link formation. Increasing the number of negatively charged phosphodiesters in the oligonucleotide increased the amount of GTG platination. Surrounding the GTG sequence with nonionic methylphosphonate linkages inhibited or eliminated cross-link formation. These observations suggest that interactions between cisplatin and the negatively charged phosphodiester backbone may play an important role in facilitating platination of guanine nucleotides in DNA.


Assuntos
Antineoplásicos/química , Cisplatino/química , Ésteres/química , Guanina/química , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Reagentes de Ligações Cruzadas/química , DNA/química , Oligodesoxirribonucleotídeos/genética , Organofosfonatos/química , Timidina Monofosfato/química
18.
Biochemistry ; 47(37): 9920-30, 2008 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-18702509

RESUMO

Interstrand cross-links (ICLs) are formed by many chemotherapeutic agents and may also arise endogenously. The mechanisms used to repair these lesions remain unclear in mammalian cells. Repair in Escherichia coli and Saccharomyces cerevisiae requires an initial unhooking step to release the tethered DNA strands. We used a panel of linear substrates containing different site-specific ICLs to characterize how structure affects ICL processing in mammalian cell extracts. We demonstrate that ICL-induced distortions affect NER-dependent and -independent processing events. The NER-dependent pathway produces dual incisions 5' to the site of the ICL as described previously [Bessho, T., et al. (1997) Mol. Cell. Biol. 17 (12), 6822-6830] but does not release the cross-link. Surprisingly, we also found that the interstrand cross-linked duplexes were unhooked in mammalian cell extracts in a manner independent of the NER pathway. Unhooking occurred identically in extracts prepared from human and rodent cells and is dependent on ATP hydrolysis and metal ions. The structure of the unhooked product was characterized and was found to contain the remnant of the cross-link. Both the NER-mediated dual 5' incisions and unhooking reactions were greatly stimulated by ICL-induced distortions, including increased local flexibility and disruption of base pairs surrounding the site of the ICL. These results suggest that in DNA not undergoing transcription or replication, distortions induced by the presence of an ICL could contribute significantly to initial cross-link recognition and processing.


Assuntos
Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Animais , Extratos Celulares , Células Cultivadas , Cricetinae , DNA/efeitos dos fármacos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Conformação de Ácido Nucleico
19.
Bioconjug Chem ; 19(5): 1064-70, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18447369

RESUMO

The study of DNA repair has been facilitated by the development of extract-based in vitro assay systems and the use of synthetic DNA duplexes that contain site-specific lesions as repair substrates. Unfortunately, exposed DNA termini can be a liability when working in crude cell extracts because they are targets for DNA end-modifying enzymes and binding sites for proteins that recognize DNA termini. In particular, the double-strand break repair protein Ku is an abundant DNA end-binding protein that has been shown to interfere with nucleotide excision repair (NER) in vitro. To facilitate the investigation of NER in whole-cell extracts, we explored ways of modifying the exposed ends of synthetic repair substrates to prevent Ku binding and improve in vitro NER efficiency. Replacement of six contiguous phosphodiester linkages at the 3'-ends of the duplex repair substrate with nuclease-resistant nonionic methylphosphonate linkages resulted in a 280-fold decrease in binding affinity between Ku and the modified duplex. These results are consistent with the published crystal structure of a Ku/DNA complex [Walker et al. (2001) Nature 412, 607-614] and show that the 3'-terminal phosphodiester linkages of linear DNA duplexes are important determinants in DNA end-binding by Ku. Using HeLa whole-cell extracts and a 149-base pair DNA duplex repair substrate, we tested the effects of modification of exposed DNA termini on NER-mediated in vitro excision of a 1,3-GTG-Pt(II) intrastrand cross-link. Methylphosphonate modification at the 3'-ends of the repair substrate resulted in a 1.6-fold increase in excision. Derivatization of the 5'-ends of the duplex with biotin and subsequent conjugation with streptavidin to block Ku binding resulted in a 2.3-fold increase excision. By combining these modifications, we were able to effectively reduce Ku-derived interference of NER excision in vitro and observed a 4.4-fold increase in platinum lesion excision. These modifications are easy to incorporate into synthetic oligonucleotides and may find general utility whenever synthetic linear duplex DNAs are used as substrates to investigate DNA repair in whole-cell extracts.


Assuntos
Reparo do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Animais , Antígenos Nucleares/química , Antígenos Nucleares/farmacologia , Sequência de Bases , Biotina/química , Extratos Celulares/química , Células Cultivadas , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/farmacologia , Células HeLa , Humanos , Autoantígeno Ku , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes/química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Compostos Organofosforados/química , Compostos Organoplatínicos/química , Homologia de Sequência do Ácido Nucleico , Ressonância de Plasmônio de Superfície , Fatores de Tempo
20.
Org Biomol Chem ; 6(22): 4212-7, 2008 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18972052

RESUMO

Triplex forming oligonucleotides (TFOs) containing the nucleoside analogues 2'-O-methyl-5-propynyluridine (1) and 2'-O-methyl-5-(3-amino-1-propynyl)uridine (2) were synthesized. The affinity and selectivity of triplex formation by these TFOs were studied by gel shift analysis, T(m) value measurement, and association rate assays. The results show that the introduction of 1 and 2 into TFOs can improve the stability of the triplexes under physiological conditions. Optimized distribution of 1 or 2 in the TFOs combined with a cluster of contiguous nucleosides with 2'-aminoethoxy sugars resulted in formation of triplexes with further enhanced stability and improved selectivity.


Assuntos
DNA/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Uridina/análogos & derivados , Uridina/química , Animais , Sequência de Bases , Cricetinae , Eletroforese em Gel de Poliacrilamida , Desnaturação de Ácido Nucleico , Oligonucleotídeos/genética , Especificidade por Substrato , Temperatura de Transição
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