RESUMO
A series of N-nitroarylated-3-chloromethyl-1,2,3,4-tetrahydroisoquinoline derivatives, several of which also possessed a trifluoromethyl substituent, were prepared and assessed as potential nitroaromatic prodrugs. The enzymatic reduction of these compounds and their cytotoxicities were studied. The compounds were cytotoxic, but this is probably not related to their enzymatic reduction.
Assuntos
Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , Nitrorredutases/antagonistas & inibidores , Pró-Fármacos/farmacologia , Tetra-Hidroisoquinolinas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/metabolismo , Nitrorredutases/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/química , Ratos , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/síntese química , Tetra-Hidroisoquinolinas/químicaRESUMO
A series of 2-nitroaryl-1,2,3,4-tetrahydroisoquinolines 10 and nitro-substituted 5,6-dihydrobenzimidazo[2,1-a]isoquinoline N-oxides 11 have been synthesised and evaluated as potential bioreducible substrates for the enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1) and Escherichia coli nitroreductase (NR). Also prepared and evaluated were 2-(3,5-dinitropyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline 12 and 5,6-dihydro-10-nitropyrido[3â³,2â³:4',5']imidazo[2',1'-a]isoquinoline 12-oxide 13. Both compounds 10b and 13 were reduced faster by human NQO1 than by CB-1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide].
Assuntos
Escherichia coli/enzimologia , Compostos Heterocíclicos/química , Isoquinolinas/química , NAD(P)H Desidrogenase (Quinona)/metabolismo , Nitrorredutases/metabolismo , Óxidos/química , Aziridinas/química , Aziridinas/farmacologia , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacologia , Humanos , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Óxidos/síntese química , Óxidos/farmacologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A series of novel DNA-interactive C8-linked pyrrolobenzodiazepine (PBD)-heterocycle polyamide conjugates has been synthesised to explore structure/sequence-selectivity relationships. One conjugate (2d) has a greater selectivity and DNA binding affinity for inverted CCAAT sequences within the Topoisomerase IIα promoter than the known C8-bis-pyrrole PBD conjugate GWL-78 (1b).
Assuntos
Amidas/química , Benzodiazepinas/química , Regiões Promotoras Genéticas , Pirróis/química , Amidas/síntese química , Amidas/metabolismo , Antígenos de Neoplasias/genética , Sequência de Bases , Benzodiazepinas/síntese química , Benzodiazepinas/metabolismo , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Regiões Promotoras Genéticas/genética , Ligação Proteica , Pirróis/síntese química , Pirróis/metabolismo , Relação Estrutura-AtividadeRESUMO
The naturally occurring pyrrolo[2,1- c][1,4]benzodiazepine (PBD) monomers such as sibiromycin, anthramycin, and tomaymycin form stable covalent adducts with duplex DNA at purine-guanine-purine sites. A correlative relationship between DNA-binding affinity, as measured by enhanced thermal denaturation temperature of calf thymus DNA ( T m), and cytotoxicity is well documented for these naturally occurring compounds and a range of synthetic analogues with sibiromycin having the highest Delta T m value (16.3 degrees C), reflecting favorable hydrogen-bonding interactions between the molecule and DNA bases. We report here that, surprisingly, the structurally simple synthetic C2-(2-naphthyl)-substituted pyrrolo[2,1- c][1,4]benzodiazepine monomer ( 5) has a Delta T m value (15.8 degrees C) similar to that of sibiromycin and significantly higher than the values for either anthramycin (13.0 degrees C) or tomaymycin (2.6 degrees C). 5 also has similar cytotoxic potency to sibiromycin which is widely regarded as the most potent naturally occurring PBD monomer. To investigate this, we have used NMR in conjunction with molecular dynamics to study the 2:1 adduct formed between 5 and the DNA duplex d(AATCTTTAAAGATT) 2. In contrast to the hydrogen-bonding interactions which predominate in the case of sibiromycin and anthramycin adducts, we have shown that the high binding affinity of 5 is due predominantly to hydrophobic (van der Waals) interactions. The high-resolution 2D NOESY, TOCSY, and COSY data obtained have also allowed unequivocal determination of the orientation of the PBD molecule (A-ring toward 3'-end of covalently bound strand), the stereochemistry at the C11 position of the PBD (C11 S), and the conformation of the C2-naphthyl ring which extends along the floor of the minor groove thus optimizing hydrophobic interactions with DNA. These results provide opportunities for future drug design in terms of extending planar hydrophobic groups at the C2 position of PBDs to maximize binding affinity.
Assuntos
Adutos de DNA/química , Oligonucleotídeos/química , Aminoglicosídeos/química , Antramicina/química , Benzodiazepinonas/química , Simulação por Computador , DNA/química , Adutos de DNA/síntese química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Oligonucleotídeos/síntese química , Temperatura de TransiçãoRESUMO
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of DNA minor groove binding agents that react covalently with guanine bases, preferably at Pu-G-Pu sites. A series of three fluorescent PBD-coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA binding affinity, cellular localization and cytotoxicity. The results show that the linker structure plays a critical role for all three parameters.
Assuntos
Benzodiazepinas/química , Benzodiazepinas/farmacologia , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , DNA/metabolismo , Pirróis/química , Pirróis/farmacologia , Benzodiazepinas/síntese química , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Microscopia de Fluorescência , Estrutura Molecular , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Pirróis/síntese química , Células Tumorais CultivadasRESUMO
Biarylpyrimidines are characterized as selective ligands for higher-order nucleic acid structures. A concise and efficient synthesis has been devised incorporating Suzuki biaryl cross-coupling of dihalopyrimidines. Two ligand series are described based on the parent thioether 4,6-bis[4-[[2-(dimethylamino)ethyl]mercapto]phenyl]pyrimidine (1a) and amide 4,6-bis(4[(2-(dimethylamino)ethyl)carboxamido]phenyl)pyrimidine (2a) compounds. In UV thermal denaturation studies with the poly(dA) x [poly(dT)]2 triplex structure, thioethers showed stabilization of the triplex form (Delta Tm < or = 20 degrees C). In contrast, amides showed duplex stabilization (Delta Tm < or = 15 degrees C) and either negligible stabilization or specific destabilization (Delta Tm = -5 degrees C) of the triplex structure. Full spectra of nucleic acid binding preferences were determined by competition dialysis. The strongest interacting thioether bound preferentially to the poly(dA) x [poly(dT)]2 triplex, K(app) = 1.6 x 10(5) M(-1) (40 x K(app) for CT DNA duplex). In contrast, the strongest binding amide selected the (T2G20T2)4 quadruplex structure, K(app) = 0.31 x 10(5) M(-1) (6.5 x K(app) for CT DNA duplex).
Assuntos
Ácidos Nucleicos/química , Pirimidinas , Telomerase/antagonistas & inibidores , Sítios de Ligação , DNA/química , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Pirimidinas/química , Pirimidinas/classificação , Pirimidinas/farmacologia , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta/métodos , Relação Estrutura-Atividade , TemperaturaRESUMO
A novel series of methyl ester-terminated C8-linked pyrrolobenzodiazepine (PBD)-poly(N-methylpyrrole) conjugates (50a-f) has been synthesized and their DNA interaction evaluated by thermal denaturation, DNA footprinting, and in vitro transcription stop assays. The synergistic effect of attaching a PBD unit to a polypyrrole fragment is illustrated by the large increase in DNA binding affinity (up to 50-fold) compared to the individual PBD and pyrrole components. 50a-f were found to bind mainly to identical DNA sequences but with apparent binding site widths increasing with molecular length and the majority of sites conforming to the consensus motif 5'-XGXWz (z = 3 +/- 1; W = A or T; X = any base but preferably a purine). They also provided robust sequence-selective blockade of transcription at sites corresponding approximately to their DNA footprints. 50a-f were shown to have good cellular/nuclear penetration properties, and a degree of correlation between cytotoxicity and DNA-binding affinity was observed.
Assuntos
Antineoplásicos/síntese química , Benzodiazepinas/síntese química , Compostos Heterocíclicos com 3 Anéis/síntese química , Pirróis/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , DNA/química , Pegada de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Modelos Moleculares , Desnaturação de Ácido Nucleico , Permeabilidade , Pirróis/química , Pirróis/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The protein O6-alkylguanine-DNA alkyltransferase (Atase) is responsible for the repair of DNA lesions generated by several clinically important anti-cancer drugs; this is manifest as active resistance in those cancer cell lines proficient in Atase expression. Novel O6-substituted guanine analogues have been synthesized, bearing acidic, basic and hydrogen bonding functional groups. In contrast to existing O6-modified purine analogues, such as methyl or benzyl, the new compounds were found to resist repair by Atase even when tested at concentrations much higher than O6-benzylguanine, a well-established Atase substrate active both in vitro and in vivo. The inactivity of the new purines as covalent substrates for Atase indicates that agents to deliver these groups to DNA would represent a new class of DNA-modifying drug that circumvents Atase-mediated resistance.
Assuntos
Enzimas Reparadoras do DNA/química , Desenho de Fármacos , Guanina/análogos & derivados , Guanina/química , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Purinas/química , Cristalografia por Raios X , DNA/química , DNA/efeitos dos fármacos , Guanina/farmacologia , Humanos , Modelos Biológicos , Estrutura Molecular , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Purinas/farmacologia , Especificidade por SubstratoRESUMO
Telomerase is a cellular ribonucleoprotein reverse transcriptase responsible for the maintenance of telomeres, the tandemly repeating guanine-rich nucleic acid sequences at the 3'-ends of eukaryotic chromosomes that serve to protect chromosomal stability and maintain integrity. Telomerase enzyme activity is essential for the sustained proliferation of most immortal cells, including cancer cells, and is currently an important recognised target for the development of novel and potentially tumour-specific anticancer chemotherapeutics. Herein, we review recent advances in the design and development of telomerase inhibitors for the treatment of cancer. To date, these have included antisense strategies, reverse transcriptase inhibitors, and agents capable of interacting with high-order telomeric DNA tetraplex (or "G-quadruplex") structures in such a way as to prevent enzyme access to its required linear telomeric DNA substrate. Critical appraisal of each distinct approach is provided together with highlighted areas for continued development necessary to further refine the present disparate classes of telomerase inhibitors for use in clinically viable therapies.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Telomerase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias/enzimologia , Telomerase/metabolismoRESUMO
A C2/C2'-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer 4b (DRG-16) with a C8-O(CH2)nO-C8' diether linkage (n = 5) has been synthesized that shows markedly superior in vitro cytotoxic potency (e.g., >3400-fold in IGROV1 ovarian cells) and interstrand DNA cross-linking reactivity (>10-fold) compared to the shorter homologue 4a (SJG-136; n = 3). In contrast, for the C-ring unsubstituted series, the corresponding n = 5 dimer (3c) is generally less cytotoxic and has a lower interstrand cross-linking reactivity compared to its shorter n = 3 homologue (3a). Dimer 4b cross-links DNA with >10-fold efficiency compared to 4a, and also inhibits the activity of the restriction endonuclease BamH1 more efficiently than either 3a or 4a. The C2-exo-unsaturated PBD dimers 4a,b are not only more effective than their C-ring saturated counterparts in terms of induced DeltaTm shift, but they also exert this effect more rapidly. Thus, while 3a and 3c exert 68 and 35% of their maximum effect immediately upon interaction with DNA, this level increases to 76 and 97% for 4a and 4b, respectively. Molecular modeling shows a rank order of 4b (n = 5) > 4a (n = 3) > 3a (n = 3) > 3c (n = 5) in terms of binding energy toward duplexes containing embedded target 5'-GAT(1-2)C cross-link sequences, reflecting the superior fit of the C2-exo-unsaturated rather than saturated C-rings of the PBD dimers. A novel synthesis of core synthetic building blocks for PBD dimers via stepwise Mitsunobu reaction and nitration with Cu(NO3)2 is also reported.
Assuntos
Antineoplásicos/síntese química , Benzodiazepinas/síntese química , Reagentes de Ligações Cruzadas/síntese química , DNA/metabolismo , Pirróis/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Autorradiografia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Desoxirribonuclease BamHI/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Ágar , Humanos , Modelos Moleculares , Desnaturação de Ácido Nucleico , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
Biarylpyrimidines bearing omega-aminoalkyl substituents have been designed as ligands for high-order DNA structures: spectrophotometric, thermal and competition equilibrium dialysis assays showed that changing the functional group for substituent attachment from thioether to amide switches the structural binding preference from triplex to tetraplex DNA; the novel ligands are non-toxic and moderate inhibitors of human telomerase.
Assuntos
DNA/química , Pirimidinas/química , Temperatura Alta , Ligantes , Estrutura Molecular , Desnaturação de Ácido NucleicoRESUMO
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of sequence-selective, DNA minor-groove binding agents that covalently attach to guanine residues. Originally derived from Streptomyces species, a number of naturally occurring PBD monomers exist with varying A-Ring and C2-substituents. One such agent, sibiromycin, is unusual in having a glycosyl residue (sibirosamine) at its A-Ring C7-position. It is the most cytotoxic member of the naturally occurring PBD family and has the highest DNA-binding affinity. Recently, the analogue 9-deoxysibiromyin was produced biosynthetically by Yonemoto and co-workers.1 Differing only in the loss of the A-Ring C9-hydroxyl group, it was reported to have a significantly higher DNA-binding affinity than sibiromycin based on DNA thermal denaturation studies, although these data have since been retracted.2 As deletion of the C9-OH moiety, which points toward the DNA minor groove floor, might intuitively be expected to reduce DNA-binding affinity through the loss of hydrogen bonding, we carried out molecular dynamics simulations on the interaction of both molecules with DNA over a 10 ns time-course in explicit solvent. Our results suggest that the two molecules may differ in their sequence-selectivity and that 9-deoxysibiromycin should have a lower binding affinity for certain sequences of DNA compared to sibiromycin. Our molecular dynamics results indicate that the C7-sibirosamine sugar does not form hydrogen bonding interactions with groups in the DNA minor-groove wall as previously reported, but instead points orthogonally out from the minor groove where it may inhibit the approach of DNA control proteins such as transcription factors. This was confirmed through a docking study involving sibiromycin and the GAL4 transcription factor, and these results could explain the significantly enhanced cytotoxicity of sibiromycin compared to other PBD family members without bulky C7-substituents.
Assuntos
Aminoglicosídeos/farmacologia , Benzodiazepinas/química , Biologia Computacional/métodos , Proteínas de Ligação a DNA/antagonistas & inibidores , DNA/química , Pirróis/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Benzodiazepinas/metabolismo , DNA/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Pirróis/metabolismo , Relação Estrutura-AtividadeRESUMO
The binding of nuclear factor Y (NF-Y) to inverted CCAAT boxes (ICBs) within the promoter region of DNA topoisomerase IIα results in control of cell differentiation and cell cycle progression. Thus, NF-Y inhibitory small molecules could be employed to inhibit the replication of cancer cells. A library of pyrrolobenzodiazepine (PBD) C8-conjugates consisting of one PBD unit attached to tri-heterocyclic polyamide fragments was designed and synthesized. The DNA-binding affinity and sequence selectivity of each compound were evaluated in DNA thermal denaturation and DNase I footprinting assays, and the ability to inhibit binding of NF-Y to ICB1 and ICB2 was studied using an electrophoretic mobility shift assay (EMSA). 3a was found to be a potent inhibitor of NF-Y binding, exhibiting a 10-fold selectivity for an ICB2 site compared to an ICB1-containing sequence, and showing low nanomolar cytotoxicity toward human tumor cell lines. Molecular modeling and computational studies have provided details of the covalent attachment process that leads to formation of the PBD-DNA adduct, and have allowed the preference of 3a for ICB2 to be rationalized.
Assuntos
Benzodiazepinas/química , DNA/metabolismo , Nylons/química , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Camundongos , Modelos Moleculares , Células NIH 3T3 , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A comprehensive SAR investigation of the C2-position of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) monomer antitumor agents is reported, establishing the molecular requirements for optimal in vitro cytotoxicity and DNA-binding affinity. Both carbocyclic and heterocyclic C2-aryl substituents have been studied ranging from single aryl rings to fused ring systems, and also styryl substituents, establishing across a library of 80 analogues that C2-aryl and styryl substituents significantly enhance both DNA-binding affinity and in vitro cytotoxicity, with a correlation between the two. The optimal C2-grouping for both DNA-binding affinity and cytotoxicity was found to be the C2-quinolinyl moiety which, according to molecular modeling, is due to the overall fit of the molecule in the DNA minor groove, and potential specific contacts with functional groups in the floor and walls of the groove. This analogue (14l) was shown to delay tumor growth in a HCT-116 (bowel) human tumor xenograft model.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Sequência de Bases , Benzodiazepinas/síntese química , Benzodiazepinas/metabolismo , Bovinos , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA/metabolismo , Feminino , Humanos , Iminas/química , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Pirróis/síntese química , Pirróis/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A 23-member C2-aryl pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione (PBD dilactam) library has been synthesized using Suzuki coupling, and the effect of base upon racemisation at the C11a-position during the cross-coupling reaction studied. Three library members (21, 30 and 33) were sufficiently cytotoxic in the NCI's preliminary screen to warrant further evaluation, and one (30, R=p-Br) was found to be cytotoxic at the sub-micromolar level in the A498 renal cancer cell line. DNA thermal denaturation studies suggested that this activity may be associated with non-covalent DNA interaction, and also demonstrated that introduction of C2-C3 unsaturation and addition of C2-aryl functionalities to the PBD dilactam skeleton significantly enhanced helix stabilisation compared to the unsubstituted PBD dilactam (6).
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzodiazepinas/síntese química , Benzodiazepinas/farmacologia , DNA de Neoplasias/metabolismo , Área Sob a Curva , Catálise , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Temperatura Alta , Humanos , Espectroscopia de Ressonância Magnética , Desnaturação de Ácido Nucleico , Espectrofotometria UltravioletaRESUMO
SJG-136 (3) is a novel pyrrolobenzodiazepine (PBD) dimer that is predicted from molecular models to bind in the minor groove of DNA and to form sequence-selective interstrand cross-links at 5'-Pu-GATC-Py-3' (Pu = purine; Py = pyrimidine) sites through covalent bonding between each PBD unit and guanines on opposing strands. Footprinting studies have confirmed that high-affinity adducts do form at 5'-G-GATC-C-3' sequences and that these can inhibit RNA polymerase in a sequence-selective manner. At higher concentrations of SJG-136, bands that migrate more slowly than one of the 5'-G-GATC-C-3' footprint sites show significantly reduced intensity, concomitant with the appearance of higher molecular weight material near the gel origin. This phenomenon is attributed to interstrand cross-linking at the 5'-G-GATC-C-3' site and is the first report of DNA footprinting being used to detect interstrand cross-linked adducts. The control dimer GD113 (4), of similar structure to SJG-136 but unable to cross-link DNA due to its C7/C7'-linkage rather than C8/C8'-linkage, neither produces footprints with the same DNA sequence nor blocks transcription at comparable concentrations. In addition to the two high-affinity 5'-G-GATC-C-3' footprints on the MS2 DNA sequence, other SJG-136 adducts of lower affinity are observed that can still block transcription but with lower efficiency. All these sites contain the 5'-GXXC-3' motif (where XX includes AG, TA, GC, CT, TT, GG, and TC) and represent less-favored cross-link sites. In time-course experiments, SJG-136 blocks transcription if incubated with a double-stranded DNA template before the transcription components are added; addition after transcription is initiated fails to elicit blockage. Single-strand ligation PCR studies on a sequence from the c-jun gene show that SJG-136 binds to 5'-GAAC-3'/5'-GTTC-3' (preferred) or 5'-GAGC-3'/5'-GCTC-3' sequences. Significantly, adducts are obtained at the same sequences following extraction of DNA from drug-treated K562 cells, confirming that the agent reaches the cellular genome and interacts with the DNA in a sequence-selective fashion. Finally, SJG-136 efficiently inhibits the action of restriction endonuclease BglII, which has a 5'-A-GATC-T-3' motif at its cleavage site.
Assuntos
Bacteriófago T7/metabolismo , Benzodiazepinonas/metabolismo , Proteínas do Capsídeo/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , DNA Viral/metabolismo , Conformação de Ácido Nucleico , Pirróis/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Bacteriófago T7/genética , Sequência de Bases , Benzodiazepinonas/farmacologia , Proteínas do Capsídeo/genética , Reagentes de Ligações Cruzadas/farmacologia , Pegada de DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , Desoxirribonuclease I/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/antagonistas & inibidores , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Pirróis/farmacologia , Proteínas de Ligação a RNA/genética , Análise de Sequência de DNA , Temperatura , Transcrição GênicaRESUMO
Molecular modelling has been carried out for a number of amine-functionalised anthraquinone derivatives to determine their extent of binding to G-tetraplex DNA and their ability to inhibit the enzymes telomerase and Taq polymerase. The results are compared to data obtained from a modified TRAP assay and show good correlation between the two methods. The findings suggest that anthraquinone derivatives of this type inhibit telomerase by stabilisation of four-stranded tetraplex structures associated with guanine-rich telomeric DNA regions.
Assuntos
Antraquinonas/química , Modelos Moleculares , Telomerase/antagonistas & inibidores , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Morte Celular/efeitos dos fármacos , DNA/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Quadruplex G , Guanina/química , Humanos , Concentração Inibidora 50 , Relação Estrutura-Atividade , Sequências de Repetição em Tandem , Taq Polimerase/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Cyclosporins are cyclic undecapeptides of fungal origin the best known of which, CsA, is a lead clinical immunosuppressant; CsG is a potential clinical immunosuppressant differing from CsA in residue 2 (L-alpha-amino-butyric acid in CsA, L-norvaline in CsG); and CsH is an inverse formyl peptide receptor agonist, differing from CsA in the chiral inversion of MeVal-11 from L to D. Crystal structure determinations of CsG and CsH were undertaken to identify structural and surface features important for biological activity and the future design of new cyclosporin derivatives. Ultra-high resolution X-ray structures (0.80 to 0.87 A resolution) determined for two crystal forms of both CsH and CsG in the presence and absence of Mg2+ are described. A major outcome of this study is the observation that the local change in chirality between CsA and CsH is associated with a major structural transformation from open beta-sheet in CsA to a highly convoluted conformation in CsH. CsG also possesses a completely novel cloverleaf motif with no H-bonded secondary structure features in spite of the minimal chemical difference with CsA. Unlike CsA, the structures of both CsH and CsG are heavily solvated. This study therefore shows that the chemical differences between the three cyclosporins, CsA, CsG and CsH can invoke unpredictably major differences in their 3D structures. The 9-11 cis-peptide bond in CsA moves to 11-1 in CsG, influencing the overall molecular conformation, while the peptide bonds in the highly convoluted loop conformation of CsH are all trans.
Assuntos
Ciclosporina/química , Cristalografia por Raios X , Ligação de Hidrogênio , Imunossupressores/química , Magnésio/química , Modelos Moleculares , Estrutura Secundária de Proteína , Receptores de Peptídeos/agonistas , Solventes/química , EstereoisomerismoRESUMO
Members of a homologous series of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers with C8-O-(CH(2))(n)-O-C8' diether linkages (n = 3-6 for 2a-d, respectively) have been studied for their ability to interact with oligonucleotide duplexes containing potential target binding sites. The results confirm earlier predictions that the n = 3 analogue (2a, DSB-120) will covalently bind to a 5'-Pu-GATC-Py sequence by cross-linking opposite-strand guanines separated by 2 bp. Preference for this DNA sequence is shown using oligonucleotides with altered bases between and/or flanking these guanines. The more extended PBD dimer 2c (n = 5) can span an extra base pair and cross-link the 5'-Pu-GA(T/A)TC-Py sequence. The ability of each homologue to cross-link linear plasmid DNA has been determined, with a rank order that correlates with the reported order of in vitro cytotoxicity: n = 3 (2a) > n = 5 (2c) > n = 6 (2d) > n = 4 (2b). The n = 3 homologue (2a) is >300-fold more efficient at cross-linking DNA than the clinically used cross-linking agent melphalan under the same conditions. Kinetic studies reveal that the n = 3 and 5 dimers achieve faster cross-linking to plasmid DNA (108 and 81% cross-linking h(-1) microM(-1) at 37 degrees C, respectively), whereas the n = 4 and 6 homologues are significantly less efficient at 10.3 and 23% cross-linking h(-1) microM(-1), respectively. Alternating activity for the odd n and even n dimers is probably due to configurational factors governed by the spatial separation of the PBD subunits and the flexible character of the tethering linkage. Molecular modeling confirms the order of cross-linking reactivity, and highlights the role of linker length in dictating sequence recognition for this class of DNA-reactive agent.
Assuntos
Benzodiazepinas/química , DNA/metabolismo , Modelos Moleculares , Pirróis/química , DNA/química , Dimerização , CinéticaRESUMO
We report examples of a series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) analogues 12-15 prepared from a common functionalized building block 11 that can be conveniently synthesized on a large scale and in optically pure form. Isoindoline analogue 15 is the most cytotoxic agent in this series, has the highest DNA-binding affinity, and shows significant activity in the in vivo hollow fibre assay.