Abstract
OBJECTIVES:
Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pe... more Abstract 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.
Thirteen compounds with diverse chemical structures have been identified as selective telomericG-... more Thirteen compounds with diverse chemical structures have been identified as selective telomericG-quadruplex-binding ligands through screening the NCI Diversity Set II, the NCI Natural Products Set II and the NCI Mechanistic Diversity Set libraries containing a total of 2,307 members against a human telomericG-quadruplex using a FRET-based melting assay. These compounds show significant selectivity towards a telomericG-quadruplex compared to duplex DNA, fall within a molecular weight range of 327–533, and are generally consistent with the Lipinski Rule of Five for drug-likeness. Thus they provide new chemicalscaffolds for the development of novel classes of G-quadruplex-targeting agents.
Pyrrolobenzodiazepine (PBD) antitumour agents have, to date, only been observed to bind to duplex... more Pyrrolobenzodiazepine (PBD) antitumour 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-NH2 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 -20oC 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 re-form. 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 hours at 50-60°C, indicating their relatively robust nature.
The pyrrolobenzodiazepines (PBDs) are covalent DNA minor-groove binding agents with a reported pr... more 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.
Abstract
Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA min... more Abstract
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-NH2 functionalities. The PBD dimer SJG-136, which has a C8–O–(CH2)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–(CH2)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.
SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinica... more 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.
Abstract
A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an effici... more Abstract A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.
We report a novel class of biaryl polyamides highly selective for G-quadruplex DNA, and with sign... more We report a novel class of biaryl polyamides highly selective for G-quadruplex DNA, and with significant cytotoxicity in several cancer cell lines; they form planar U-shaped structures that match the surface area dimensions of a terminal G-quartet in quadruplex structures rather than the grooves of duplex DNA.
Pyrrolobenzodiazepines (PBDs) are sequence-selective DNA minor-groove binding agents that covalen... more 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.
Abstract
Objectives The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple add... more Abstract
Objectives The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA.
Methods Formation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis.
Results ELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway.
Conclusions The data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.
A dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex DNA adducts of a pyrrolobenzodi... more A dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex DNA adducts of a pyrrolobenzodiazepine (PBD) minor groove binding agent (1) 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.
A 30 second burst of microwave irradiation at an energy level insufficient to cause DNA denaturat... more A 30 second burst of microwave irradiation at an energy level insufficient to cause DNA denaturation or damage drives the covalent reaction between pyrrolobenzodiazepine (PBD) antitumour agents and double-stranded or hairpin oligonucleotides to completion, a process that normally takes between 3–24 hours and thus offering the opportunity for higher-throughput screening of covalent-binding DNA-interactive agents.
Abstract:
The binding of betamethasone sodium phosphate and prednisolone, two steroidal antiinfla... more Abstract: The binding of betamethasone sodium phosphate and prednisolone, two steroidal antiinflammatory drugs and theophylline sodium glycinate, a bronchodilator, to bovine serum albumin (BSA), has been studied by equilibrium dialysis (ED) method at different temperature and pH values for characterizing the binding of these drugs to BSA. Binding was exothermic, entropically driven and spontaneous, as indicated by the thermodynamic analysis. The major part of the binding energy at site II results from electrostatic and hydrophobic interactions. The free fraction of either betamethasone or prednisolone in the presence of theophylline sodium glycinate and vice versa was monitored in the presence and absence of site specific probes. The free fraction of betamethasone sodium phosphate by theophylline sodium glycinate and vice versa was increased during concurrent administration causing reduced binding of these drugs to BSA. This increment of free fraction was more prominent in the presence of site I specific probe, which suggested that in the absence of site I specific probe, betamethasone after being displaced by theophylline from its high affinity site rebound to its low affinity site. Similar type of result was observed in case of prednisolonetheophylline interaction.
Abstract
Two quinones, stereochenols A (1) and B (2) were isolated from a methanol extract of the... more Abstract Two quinones, stereochenols A (1) and B (2) were isolated from a methanol extract of the stem bark of Stereospermum chelonoides, in addition to the known naphthoquinones, sterekunthal B (3) and sterequinone C (4). The structures of these compounds were established by extensive spectroscopic analyses and by comparison of their spectral data with those of related compounds.
Abstract
A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fra... more Abstract A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fraction comprising arborinine and γ-fagarine (VLC), showed mild to significant in vitro antibacterial activity. In a brine shrimp lethality bioassay, the extract and the fraction were found to exhibit moderate cytotoxicity having LC50 of 76.26 μg/ml and 14.98 μg/ml, respectively.
Abstract
The cytotoxicity and antibacterial activity of petroleum ether, chloroform and methanol ... more Abstract The cytotoxicity and antibacterial activity of petroleum ether, chloroform and methanol extracts of Wedelia calendulacea were assayed by brine shrimp lethality bioassay and standardized disk diffusion method against 19 bacterial strains. Three diterpenes isolated from the plant were also evaluated for in vitro antibacterial activities. The LC50 for the crude extracts against the brine shrimp nauplii were found to be 4.59 microg/ml, 7.99 microg/ml and 14.88 microg/ml, respectively, whereas the positive control, vincristine sulfate showed an LC50 of 0.58 microg/ml. Among the crude extracts and pure compounds tested, (-)-kaur-16-en-19-oic acid isolated from the chloroform extract showed the highest inhibitory activity against most of the bacterial strains with mean zone of inhibition of 10-21 mm at 200 microg/disc.
Abstract
The antitumor activity of the ethanolic extracts of 12 medicinal plants of Bangladesh, i... more Abstract The antitumor activity of the ethanolic extracts of 12 medicinal plants of Bangladesh, including the vincristine–vinblastine producing Catharanthus roseus was studied using the potato disk bioassay technique. Among these, 10 plant extracts at 25.0-μg/disc exhibited significant inhibition of crown gall tumors caused by Agrobacterium tumefaciens.
Abstract
OBJECTIVES:
Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pe... more Abstract 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.
Thirteen compounds with diverse chemical structures have been identified as selective telomericG-... more Thirteen compounds with diverse chemical structures have been identified as selective telomericG-quadruplex-binding ligands through screening the NCI Diversity Set II, the NCI Natural Products Set II and the NCI Mechanistic Diversity Set libraries containing a total of 2,307 members against a human telomericG-quadruplex using a FRET-based melting assay. These compounds show significant selectivity towards a telomericG-quadruplex compared to duplex DNA, fall within a molecular weight range of 327–533, and are generally consistent with the Lipinski Rule of Five for drug-likeness. Thus they provide new chemicalscaffolds for the development of novel classes of G-quadruplex-targeting agents.
Pyrrolobenzodiazepine (PBD) antitumour agents have, to date, only been observed to bind to duplex... more Pyrrolobenzodiazepine (PBD) antitumour 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-NH2 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 -20oC 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 re-form. 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 hours at 50-60°C, indicating their relatively robust nature.
The pyrrolobenzodiazepines (PBDs) are covalent DNA minor-groove binding agents with a reported pr... more 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.
Abstract
Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA min... more Abstract
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-NH2 functionalities. The PBD dimer SJG-136, which has a C8–O–(CH2)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–(CH2)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.
SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinica... more 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.
Abstract
A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an effici... more Abstract A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.
We report a novel class of biaryl polyamides highly selective for G-quadruplex DNA, and with sign... more We report a novel class of biaryl polyamides highly selective for G-quadruplex DNA, and with significant cytotoxicity in several cancer cell lines; they form planar U-shaped structures that match the surface area dimensions of a terminal G-quartet in quadruplex structures rather than the grooves of duplex DNA.
Pyrrolobenzodiazepines (PBDs) are sequence-selective DNA minor-groove binding agents that covalen... more 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.
Abstract
Objectives The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple add... more Abstract
Objectives The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA.
Methods Formation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis.
Results ELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway.
Conclusions The data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.
A dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex DNA adducts of a pyrrolobenzodi... more A dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex DNA adducts of a pyrrolobenzodiazepine (PBD) minor groove binding agent (1) 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.
A 30 second burst of microwave irradiation at an energy level insufficient to cause DNA denaturat... more A 30 second burst of microwave irradiation at an energy level insufficient to cause DNA denaturation or damage drives the covalent reaction between pyrrolobenzodiazepine (PBD) antitumour agents and double-stranded or hairpin oligonucleotides to completion, a process that normally takes between 3–24 hours and thus offering the opportunity for higher-throughput screening of covalent-binding DNA-interactive agents.
Abstract:
The binding of betamethasone sodium phosphate and prednisolone, two steroidal antiinfla... more Abstract: The binding of betamethasone sodium phosphate and prednisolone, two steroidal antiinflammatory drugs and theophylline sodium glycinate, a bronchodilator, to bovine serum albumin (BSA), has been studied by equilibrium dialysis (ED) method at different temperature and pH values for characterizing the binding of these drugs to BSA. Binding was exothermic, entropically driven and spontaneous, as indicated by the thermodynamic analysis. The major part of the binding energy at site II results from electrostatic and hydrophobic interactions. The free fraction of either betamethasone or prednisolone in the presence of theophylline sodium glycinate and vice versa was monitored in the presence and absence of site specific probes. The free fraction of betamethasone sodium phosphate by theophylline sodium glycinate and vice versa was increased during concurrent administration causing reduced binding of these drugs to BSA. This increment of free fraction was more prominent in the presence of site I specific probe, which suggested that in the absence of site I specific probe, betamethasone after being displaced by theophylline from its high affinity site rebound to its low affinity site. Similar type of result was observed in case of prednisolonetheophylline interaction.
Abstract
Two quinones, stereochenols A (1) and B (2) were isolated from a methanol extract of the... more Abstract Two quinones, stereochenols A (1) and B (2) were isolated from a methanol extract of the stem bark of Stereospermum chelonoides, in addition to the known naphthoquinones, sterekunthal B (3) and sterequinone C (4). The structures of these compounds were established by extensive spectroscopic analyses and by comparison of their spectral data with those of related compounds.
Abstract
A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fra... more Abstract A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fraction comprising arborinine and γ-fagarine (VLC), showed mild to significant in vitro antibacterial activity. In a brine shrimp lethality bioassay, the extract and the fraction were found to exhibit moderate cytotoxicity having LC50 of 76.26 μg/ml and 14.98 μg/ml, respectively.
Abstract
The cytotoxicity and antibacterial activity of petroleum ether, chloroform and methanol ... more Abstract The cytotoxicity and antibacterial activity of petroleum ether, chloroform and methanol extracts of Wedelia calendulacea were assayed by brine shrimp lethality bioassay and standardized disk diffusion method against 19 bacterial strains. Three diterpenes isolated from the plant were also evaluated for in vitro antibacterial activities. The LC50 for the crude extracts against the brine shrimp nauplii were found to be 4.59 microg/ml, 7.99 microg/ml and 14.88 microg/ml, respectively, whereas the positive control, vincristine sulfate showed an LC50 of 0.58 microg/ml. Among the crude extracts and pure compounds tested, (-)-kaur-16-en-19-oic acid isolated from the chloroform extract showed the highest inhibitory activity against most of the bacterial strains with mean zone of inhibition of 10-21 mm at 200 microg/disc.
Abstract
The antitumor activity of the ethanolic extracts of 12 medicinal plants of Bangladesh, i... more Abstract The antitumor activity of the ethanolic extracts of 12 medicinal plants of Bangladesh, including the vincristine–vinblastine producing Catharanthus roseus was studied using the potato disk bioassay technique. Among these, 10 plant extracts at 25.0-μg/disc exhibited significant inhibition of crown gall tumors caused by Agrobacterium tumefaciens.
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Papers by Khondaker Miraz Rahman
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.
Keywords: Pyrrolo[2,1-c][1,4]benzodiazepine (PBD); antitumor agents; GWL-78; DNA; minor-groove binder
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-NH2 functionalities. The PBD dimer SJG-136, which has a C8–O–(CH2)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–(CH2)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.
A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.
Objectives The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA.
Methods Formation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis.
Results ELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway.
Conclusions The data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.
The binding of betamethasone sodium phosphate and prednisolone, two steroidal antiinflammatory drugs and theophylline sodium glycinate, a bronchodilator, to bovine serum albumin (BSA), has been studied by equilibrium dialysis (ED) method at different temperature and pH values for characterizing the binding of these drugs to BSA. Binding was exothermic, entropically driven and spontaneous, as indicated by the thermodynamic analysis. The major part of the binding energy at site II results from electrostatic and hydrophobic interactions. The free fraction of either betamethasone or prednisolone in the presence of theophylline sodium glycinate and vice versa was monitored in the presence and absence of site specific probes. The free fraction of betamethasone sodium phosphate by theophylline sodium glycinate and vice versa was increased during concurrent administration causing reduced binding of these drugs to BSA. This increment of free fraction was more prominent in the presence of site I specific probe, which suggested that in the absence of site I specific probe, betamethasone after being displaced by theophylline from its high affinity site rebound to its low affinity site. Similar type of result was observed in case of prednisolonetheophylline interaction.
Two quinones, stereochenols A (1) and B (2) were isolated from a methanol extract of the stem bark of Stereospermum chelonoides, in addition to the known naphthoquinones, sterekunthal B (3) and sterequinone C (4). The structures of these compounds were established by extensive spectroscopic analyses and by comparison of their spectral data with those of related compounds.
A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fraction comprising arborinine and γ-fagarine (VLC), showed mild to significant in vitro antibacterial activity. In a brine shrimp lethality bioassay, the extract and the fraction were found to exhibit moderate cytotoxicity having LC50 of 76.26 μg/ml and 14.98 μg/ml, respectively.
Keywords: Ravenia spectabilis; Antibacterial; Cytotoxicity
The cytotoxicity and antibacterial activity of petroleum ether, chloroform and methanol extracts of Wedelia calendulacea were assayed by brine shrimp lethality bioassay and standardized disk diffusion method against 19 bacterial strains. Three diterpenes isolated from the plant were also evaluated for in vitro antibacterial activities. The LC50 for the crude extracts against the brine shrimp nauplii were found to be 4.59 microg/ml, 7.99 microg/ml and 14.88 microg/ml, respectively, whereas the positive control, vincristine sulfate showed an LC50 of 0.58 microg/ml. Among the crude extracts and pure compounds tested, (-)-kaur-16-en-19-oic acid isolated from the chloroform extract showed the highest inhibitory activity against most of the bacterial strains with mean zone of inhibition of 10-21 mm at 200 microg/disc.
The antitumor activity of the ethanolic extracts of 12 medicinal plants of Bangladesh, including the vincristine–vinblastine producing Catharanthus roseus was studied using the potato disk bioassay technique. Among these, 10 plant extracts at 25.0-μg/disc exhibited significant inhibition of crown gall tumors caused by Agrobacterium tumefaciens.
Keywords: Medicinal plants; Bangladesh; Antitumor activity
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.
Keywords: Pyrrolo[2,1-c][1,4]benzodiazepine (PBD); antitumor agents; GWL-78; DNA; minor-groove binder
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-NH2 functionalities. The PBD dimer SJG-136, which has a C8–O–(CH2)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–(CH2)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.
A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.
Objectives The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA.
Methods Formation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis.
Results ELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway.
Conclusions The data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.
The binding of betamethasone sodium phosphate and prednisolone, two steroidal antiinflammatory drugs and theophylline sodium glycinate, a bronchodilator, to bovine serum albumin (BSA), has been studied by equilibrium dialysis (ED) method at different temperature and pH values for characterizing the binding of these drugs to BSA. Binding was exothermic, entropically driven and spontaneous, as indicated by the thermodynamic analysis. The major part of the binding energy at site II results from electrostatic and hydrophobic interactions. The free fraction of either betamethasone or prednisolone in the presence of theophylline sodium glycinate and vice versa was monitored in the presence and absence of site specific probes. The free fraction of betamethasone sodium phosphate by theophylline sodium glycinate and vice versa was increased during concurrent administration causing reduced binding of these drugs to BSA. This increment of free fraction was more prominent in the presence of site I specific probe, which suggested that in the absence of site I specific probe, betamethasone after being displaced by theophylline from its high affinity site rebound to its low affinity site. Similar type of result was observed in case of prednisolonetheophylline interaction.
Two quinones, stereochenols A (1) and B (2) were isolated from a methanol extract of the stem bark of Stereospermum chelonoides, in addition to the known naphthoquinones, sterekunthal B (3) and sterequinone C (4). The structures of these compounds were established by extensive spectroscopic analyses and by comparison of their spectral data with those of related compounds.
A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fraction comprising arborinine and γ-fagarine (VLC), showed mild to significant in vitro antibacterial activity. In a brine shrimp lethality bioassay, the extract and the fraction were found to exhibit moderate cytotoxicity having LC50 of 76.26 μg/ml and 14.98 μg/ml, respectively.
Keywords: Ravenia spectabilis; Antibacterial; Cytotoxicity
The cytotoxicity and antibacterial activity of petroleum ether, chloroform and methanol extracts of Wedelia calendulacea were assayed by brine shrimp lethality bioassay and standardized disk diffusion method against 19 bacterial strains. Three diterpenes isolated from the plant were also evaluated for in vitro antibacterial activities. The LC50 for the crude extracts against the brine shrimp nauplii were found to be 4.59 microg/ml, 7.99 microg/ml and 14.88 microg/ml, respectively, whereas the positive control, vincristine sulfate showed an LC50 of 0.58 microg/ml. Among the crude extracts and pure compounds tested, (-)-kaur-16-en-19-oic acid isolated from the chloroform extract showed the highest inhibitory activity against most of the bacterial strains with mean zone of inhibition of 10-21 mm at 200 microg/disc.
The antitumor activity of the ethanolic extracts of 12 medicinal plants of Bangladesh, including the vincristine–vinblastine producing Catharanthus roseus was studied using the potato disk bioassay technique. Among these, 10 plant extracts at 25.0-μg/disc exhibited significant inhibition of crown gall tumors caused by Agrobacterium tumefaciens.
Keywords: Medicinal plants; Bangladesh; Antitumor activity