RESUMO
The pyrrolobenzodiazepines (PBDs) are a family of covalent-binding DNA-interactive minor-groove binding agents with a thermodynamic preference for binding to 5'-Pu-G-Pu-3' sequences (Pu = Purine) but a kinetic preference for 5'-Py-G-Py-3' (Py = Pyrimidine). Using HPLC/MS methodology and a range of designed hairpin-forming oligonucleotides, the kinetics of reaction of a C8-bis-pyrrole pyrrolobenzodiazepine (PBD) conjugate (GWL-78, 2) with sixteen isomeric oligonucleotides has been evaluated, each containing a single PBD binding site in one of two locations. The PBD-binding base-pair triplets were designed to include every possible combination of A and T bases adjacent to the covalently-reacting guanine, with the set of hairpins consisting of isomeric pairs containing the same sequence in the hairpin stem but with either hexaethylene glycol (HEG) or TTT loops. The PBD 2 reacted most rapidly with TGT and TGA sequences, with the possibility that adducts might form in both the 3'- and 5'-directions with some sequences according to modelling studies. A faster reaction rate was observed for all hairpins containing the HEG loop except one (Seq 10) when the PBD binding triplets were located either near the loop or adjacent to the 5'-end. Modelling studies have suggested that this difference in reactivity could be due to the structural flexibility of the HEG loop allowing both A-ring-3' and A-ring-5' adducts to form, while a TTT loop should favour only A-ring-5' adducts due to steric considerations. These findings contrast with the results reported by Nguyen and Wilson for the interaction of non-covalent DNA-binding molecules with DNA hairpins, where the loop structure was found to have little effect on interaction in the main stem of the hairpin.
Assuntos
Antineoplásicos/metabolismo , Benzodiazepinas/metabolismo , Adutos de DNA/química , Adutos de DNA/genética , Dipeptídeos/metabolismo , Sequências Repetidas Invertidas , Pareamento de Bases , Sequência de Bases , Adutos de DNA/metabolismo , Modelos MolecularesRESUMO
The alarming rise of extensively drug-resistant tuberculosis (XDR-TB) strains, compel the development of new molecules with novel modes of action to control this world health emergency. Distamycin analogues containing N-terminal biaryl-motifs 2(1-5)(1-7) were synthesised using a solution-phase approach and evaluated for their anti-mycobacterial activity and DNA-sequence selectivity. Thiophene dimer motif-containing polyamide 2(2,6) exhibited 10-fold higher inhibitory activity against Mycobacterium tuberculosis compared to distamycin and library member 2(5,7) showed high binding affinity for the 5'-ACATAT-3' sequence.
Assuntos
Antituberculosos/síntese química , DNA Bacteriano/antagonistas & inibidores , Distamicinas/síntese química , Nylons/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Antituberculosos/farmacologia , Sítios de Ligação , Técnicas de Química Combinatória , Pegada de DNA , DNA Bacteriano/química , Distamicinas/farmacologia , Ligantes , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Nylons/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tiofenos/químicaRESUMO
Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents that possess two electrophilic imine moieties (or their equivalent) capable of forming covalent aminal linkages with guanine C2-NH(2) functionalities. The PBD dimer SJG-136, which has a C8-O-(CH(2))(3)-O-C8'' central linker joining the two PBD moieties, is currently undergoing phase II clinical trials and current research is focused on developing analogues of SJG-136 with different linker lengths and substitution patterns. Using a reversed-phase ion pair HPLC/MS method to evaluate interaction with oligonucleotides of varying length and sequence, we recently reported (JACS, 2009, 131, 13 756) that SJG-136 can form three different types of adducts: inter- and intrastrand cross-linked adducts, and mono-alkylated adducts. These studies have now been extended to include PBD dimers with a longer central linker (C8-O-(CH(2))(5)-O-C8'), demonstrating that the type and distribution of adducts appear to depend on (i) the length of the C8/C8'-linker connecting the two PBD units, (ii) the positioning of the two reactive guanine bases on the same or opposite strands, and (iii) their separation (i.e. the number of base pairs, usually ATs, between them). Based on these data, a set of rules are emerging that can be used to predict the DNA-interaction behaviour of a PBD dimer of particular C8-C8' linker length towards a given DNA sequence. These observations suggest that it may be possible to design PBD dimers to target specific DNA sequences.
Assuntos
Benzodiazepinas/química , Reagentes de Ligações Cruzadas/química , DNA/química , Pirróis/química , Sequência de Bases , Benzodiazepinonas/química , Cromatografia Líquida de Alta Pressão , Dimerização , Espectrometria de Massas , Modelos Moleculares , Oligonucleotídeos/químicaRESUMO
OBJECTIVES: Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pentyldioxy linkers, possess potent bactericidal activity against a range of Gram-positive bacteria by virtue of their capacity to cross-link duplex DNA in sequence-selective fashion. Here we attempt to improve the antibacterial activity and cytotoxicity profile of PBD-containing conjugates by extension of dimer linkers and replacement of one PBD unit with phenyl-substituted or benzo-fused heterocycles that facilitate non-covalent interactions with duplex DNA. METHODS: DNase I footprinting was used to identify high-affinity DNA binding sites. A staphylococcal gene microarray was used to assess epidemic methicillin-resistant Staphylococcus aureus 16 phenotypes induced by PBD conjugates. Molecular dynamics simulations were employed to investigate the accommodation of compounds within the DNA helix. RESULTS: Increasing the length of the linker in PBD dimers led to a progressive reduction in antibacterial activity, but not in their cytotoxic capacity. Complex patterns of DNA binding were noted for extended PBD dimers. Modelling of DNA strand cross-linking by PBD dimers indicated distortion of the helix. A majority (26 of 43) of PBD-biaryl conjugates possessed potent antibacterial activity with little or no helical distortion and a more favourable cytotoxicity profile. Bactericidal activity of PBD-biaryl conjugates was determined by inability to excise covalently bound drug molecules from bacterial duplex DNA. CONCLUSIONS: PBD-biaryl conjugates have a superior antibacterial profile compared with PBD dimers such as ELB-21. We have identified six PBD-biaryl conjugates as potential drug development candidates.
Assuntos
Antibacterianos/farmacologia , Benzodiazepinas/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pirróis/farmacologia , Antibacterianos/metabolismo , Benzodiazepinas/metabolismo , Sítios de Ligação , Pegada de DNA , DNA Bacteriano/metabolismo , Perfilação da Expressão Gênica , Análise em Microsséries , Viabilidade Microbiana/efeitos dos fármacos , Simulação de Dinâmica Molecular , Pirróis/metabolismoRESUMO
Pyrrolobenzodiazepine (PBD) antitumor agents have, to date, only been observed to bind to duplex DNA, apparently requiring a minor groove environment for covalent bond formation between their C11-position and the C2-NH(2) functionality of a guanine base. Using an HPLC/MS assay we have now observed and isolated for the first time PBD adducts with single-stranded DNA fragments. Surprisingly, these adducts could only be formed through dissociation of duplex DNA adducts and not by direct interaction of PBDs with single-stranded DNA. They were sufficiently stable for characterization by MALDI-TOF-MS and remained intact after storing at -20 °C for at least 20 days, although the PBD became detached from the DNA within 7 days if stored at room temperature. Furthermore, addition of a complementary strand allowed the duplex adduct to reform. The relative stability of single-stranded PBD/DNA adducts despite a complete loss of minor groove structure was further confirmed by CD spectroscopic analysis. The CD signal induced by the presence of a PBD molecule in the single-stranded adducts remained prominent despite heating for 2 h at 50-60 °C, thus indicating their relatively robust nature.
Assuntos
Antineoplásicos/análise , Benzodiazepinas/análise , Adutos de DNA/análise , DNA de Cadeia Simples/metabolismo , Pirróis/análise , Antineoplásicos/farmacologia , Sequência de Bases , Benzodiazepinas/farmacologia , Adutos de DNA/metabolismo , DNA de Cadeia Simples/química , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , Pirróis/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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
Pyrrolobenzodiazepines (PBDs) are sequence-selective DNA minor-groove binding agents that covalently bond to guanine with a reported preference for Pu-G-Pu sequences (Pu = Purine). Using HPLC/MS and Circular Dichroism (CD) methodologies, we have established for the first time that the aminal bond formed between PBD molecules and DNA is reversible. Furthermore, we have shown that while the rate of aminal bond cleavage does not depend on the sequence preference of a PBD molecule for a particular binding site, the rate of re-formation of the PBD-DNA adduct does. We have also shown that the PBD anthramycin (2) appears to be an exception to this rule in that, during cleavage from the DNA, its C-ring aromatizes and it cannot then re-attach due to a loss of electrophilicity at the C11-position. Although the C-ring aromatization of anthramycin has been previously reported to occur in the absence of DNA and after treatment with trifluoroacetic acid (TFA), in this case no pH lowering was required, with the DNA itself appearing to catalyse the process.
Assuntos
Benzodiazepinas/química , Adutos de DNA/química , Pirróis/química , Catálise , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Modelos Moleculares , Estrutura MolecularRESUMO
SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials. Using a HPLC/MS-based methodology developed to evaluate the binding of DNA-interactive agents to oligonucleotides of varying length and sequence, we have demonstrated that, in addition to the previously known interstrand cross-link at Pu-GATC-Py sequences, 1 can form a longer interstrand cross-link at Pu-GAATC-Py sequences, an intrastrand cross-link at both shorter Pu-GATG-Py and longer Pu-GAATG-Py sequences, and, in addition, monoalkylated adducts at suitable PBD binding sites where neither intra- or interstrand cross-links are feasible because of the unavailability of two appropriately positioned guanines. Crucially, we have demonstrated a preference for the extended intrastrand cross-link with Pu-GAATG-Py, which forms more rapidly than the other cross-links (rank order: Pu-GAATG-Py > Pu-GATC-Py >> Pu-GATG-Py and Pu-GAATC-Py). However, thermal denaturation studies suggest that the originally reported Pu-GATC-Py interstrand cross-link is more stable, consistent with the covalent joining of both strands of the duplex and a lower overall distortion of the helix according to modeling studies. These observations impact on the proposed mechanism of action of SJG-136 (1) both in vitro and in vivo, the repair of its adducts and mechanism of resistance in cells, and potentially on the type of pharmacodynamic assay used in clinical trials.
Assuntos
Benzodiazepinonas/química , Reagentes de Ligações Cruzadas/química , Adutos de DNA/química , Pirróis/química , Sequência de Bases , Dimerização , Conformação de Ácido NucleicoRESUMO
A dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex DNA adducts of a pyrrolobenzodiazepine (PBD) minor groove binding agent () has been observed for the first time. The equilibrium, which establishes over 1 hour and must require unfolding of both types of adducts, is surprising given that PBDs normally require DNA minor groove structure for binding and take 24 hours for complete reaction with duplex DNA. The equilibrium is interesting from an energetics perspective due to the well known DNA stabilizing effect of PBDs. This observation could have significance for the in vitro and in vivo biological activity of PBDs, as DNA hairpin and loop structures are known to be important in cellular processes such as transcription and replication.
Assuntos
Benzodiazepinas/farmacologia , Adutos de DNA/química , Adutos de DNA/efeitos dos fármacos , DNA/química , DNA/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Pirróis/farmacologia , Sequência de Bases , Benzodiazepinas/química , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Pirróis/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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
DNA binding 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) building blocks have been developed that span two DNA base pairs with a strong preference for GC-rich DNA. They have been conjugated to a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) molecule to produce C8-linked PBD-MPB hybrids that can stabilize GC-rich DNA by up to 13-fold compared to AT-rich DNA. Some have subpicomolar IC50 values in human tumor cell lines and in primary chronic lymphocytic leukemia cells, while being up to 6 orders less cytotoxic in the non-tumor cell line WI38, suggesting that key DNA sequences may be relevant targets in these ultrasensitive cancer cell lines. One conjugate, 7h (KMR-28-39), which has femtomolar activity in the breast cancer cell line MDA-MB-231, has significant dose-dependent antitumor activity in MDA-MB-231 (breast) and MIA PaCa-2 (pancreatic) human tumor xenograft mouse models with insignificant toxicity at therapeutic doses. Preliminary studies suggest that 7h may sterically inhibit interaction of the transcription factor NF-κB with its cognate DNA binding sequence.
Assuntos
Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , Sequência Rica em GC , Animais , Benzodiazepinas/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Transferência Ressonante de Energia de Fluorescência , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Estrutura Molecular , NF-kappa B/antagonistas & inibidores , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The pyrrolobenzodiazepines (PBDs) are covalent DNA minor-groove binding agents with a reported preference for binding to 5'-Pu-G-Pu sequences with their A rings oriented toward the 3'-end of the covalently modified DNA strand. Using HPLC/MS methodology and a range of designed hairpin-forming 17-mer oligonucleotides, the kinetics of reaction of a bis-pyrrole PBD conjugate (GWL-78, 2) has been evaluated with eight isomeric oligonucleotides, each containing a single PBD binding site in one of two locations. The PBD-binding base pair triplets were designed to include every possible combination of A and T bases adjacent to the covalently reacting guanine. Contrary to expectations, 2 reacted most rapidly with TGT and TGA sequences, and adducts were observed to form in both the 3'- and the 5'-directions. Molecular modeling studies revealed that for 3'-oriented adducts, this preference could be explained by formation of a hydrogen bond between the N10-H of the PBD and the oxygen of the C2-carbonyl of a thymine base on the 3'-side of the covalently bound guanine. For 5'-adducts, an analogous PBD N10-H hydrogen bond may form instead to the N3 of an equivalent adenine on the opposite strand.
RESUMO
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
The azinomycins are potent antitumour antibiotics that are able to crosslink DNA, but are relatively unstable and unlikely to progress as therapeutic candidates. A prototype analogue 4 with more clinical potential has been designed and synthesised and incorporates the epoxide function of the azinomycins and a nitrogen mustard. Two further analogues 5 and 6 that can alkylate DNA but cannot crosslink the duplex have also been synthesised. Compound 4 crosslinks DNA efficiently at nM concentrations. Compounds 4-6 were submitted to the NCI 60 cell line screen and have similar antitumour activity, although 4 is slightly less active than the non-crosslinking compounds. These observations will be important in the design of further azinomycin analogues with antitumour activity.