Isolation, chemistry, and biology of pyrrolo[1,4]benzodiazepine natural products.

The isolation of the antitumor antibiotic anthramycin in the 1960s prompted extensive research into pyrrolo[1,4]benzodiazepines (PBD) as potential therapeutics for the treatment of cancers. Since then, nearly 60 PBD natural products have been isolated and evaluated with regard to their biological activity. Synthetic studies and total syntheses have enabled access to PBD analogues, culminating in the development of highly potent anticancer agents. This review provides a summary of the occurrence and biological activity of PBD natural products and covers the strategies employed for their total syntheses.

Topical delivery of anthramycin II. Influence of binary and ternary solvent systems.

Anthramycin (ANT) is a member of the pyrolobenzodiazepine family and is a potent cytotoxic agent. Previously, we reported the topical delivery of ANT from a range of solvents that may also act as skin penetration enhancers (SPEs). The skin penetration and uptake was monitored for simple solutions of ANT in propylene glycol (PG), dipropylene glycol (DiPG), Transcutol P (TC), isopropyl myristate (IPM), propylene glycol monocaprylate (PGMC) and propylene glycol monolaurate (PGML). The amounts of PG, DiPG and TC that were taken up by, and that penetrated the skin were also measured, with a clear dependence of ANT penetration on the rate and extent of PG and TC permeation. The present work investigates ANT skin delivery from a range of binary and ternary systems to determine any potential improvement in skin uptake compared with earlier results for the neat solvents. Following miscibility and stability studies a total of eight formulations were taken forward for evaluation in human skin in vitro. Binary systems of PG and water did not result in any skin permeation of ANT. Combining PG with either PGMC or PGML did promote skin penetration of ANT but no significant improvement was evident compared with PG alone. More complex ternary systems based on PG, DiPG, PGMC, PGML and water also did not show significant improvements on ANT permeation, compared with single solvents. Total skin penetration and retention of ANT ranged from 1 to 6% across all formulations studied. Where ANT was delivered to the receptor phase there were also high amounts of PG permeation with >50% and ~35% PG present for the binary systems and ternary vehicles, respectively. These findings along with our previous paper confirm PG as a suitable solvent / SPE for ANT either alone or in combination with PGML or PGMC. The results also underline the necessity for empirical testing to determine whether or not a vehicle is acting as a SPE for a specific active in a topical formulation.

Sequence-selective binding of C8-conjugated pyrrolobenzodiazepines (PBDs) to DNA.

DNA footprinting and melting experiments have been used to examine the sequence-specific binding of C8-conjugates of pyrrolobenzodiazepines (PBDs) and benzofused rings including benzothiophene and benzofuran, which are attached using pyrrole- or imidazole-containing linkers. The conjugates modulate the covalent attachment points of the PBDs, so that they bind best to guanines flanked by A/T-rich sequences on either the 5'- or 3'-side. The linker affects the binding, and pyrrole produces larger changes than imidazole. Melting studies with 14-mer oligonucleotide duplexes confirm covalent attachment of the conjugates, which show a different selectivity to anthramycin and reveal that more than one ligand molecule can bind to each duplex.

Topical delivery of anthramycin I. Influence of neat solvents.

Anthramycin (ANT) was the first pyrrolobenzodiazepine (PBD) molecule to be isolated, and is a potent cytotoxic agent. Although the PBD family has been investigated for use in systemic chemotherapy, their application in the management of actinic keratoses (AK) or skin cancer has not been investigated to date. In the present work, anthramycin (ANT) was selected as a model PBD compound, and the skin penetration of the molecule was investigated using conventional Franz diffusion cells. Finite dose permeation studies of ANT were performed using propylene glycol (PG), 1,3-butanediol (BD), dipropylene glycol (DiPG), Transcutol P® (TC), propylene glycol monocaprylate (PGMC), propylene glycol monolaurate (PGML) and isopropyl myristate (IPM). The skin penetration of BD, DiPG, PG and TC was also measured. Penetration of ANT through human skin was evident for TC, PG and PGML with the active appearing to "track" the permeation of the vehicle in the case of TC and PG. Deposition of ANT in skin could be correlated with skin retention of the vehicle in the case of IPM, PGMC and PGML. These preliminary findings confirm the ability of ANT to penetrate human skin and, given the potency of the molecule, suggest that further investigation is justified. Additionally, the findings emphasise the critical importance of understanding the fate of the excipient for the rational design of topical formulations.

From Anthramycin to Pyrrolobenzodiazepine (PBD)-Containing Antibody-Drug Conjugates (ADCs).

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of sequence-selective DNA minor-groove binding agents that form a covalent aminal bond between their C11-position and the C2-NH2 groups of guanine bases. The first example of a PBD monomer, the natural product anthramycin, was discovered in the 1960s, and the best known PBD dimer, SJG-136 (also known as SG2000, NSC 694501 or BN2629), was synthesized in the 1990s and has recently completed Phase II clinical trials in patients with leukaemia and ovarian cancer. More recently, PBD dimer analogues are being attached to tumor-targeting antibodies to create antibody-drug conjugates (ADCs), a number of which are now in clinical trials, with many others in pre-clinical development. This Review maps the development from anthramycin to the first PBD dimers, and then to PBD-containing ADCs, and explores both structure-activity relationships (SARs) and the biology of PBDs, and the strategies for their use as payloads for ADCs.

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments.

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.

Identification of the dioxygenase-generated intermediate formed during biosynthesis of the dihydropyrrole moiety common to anthramycin and sibiromycin.

A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis is a prerequisite for engineering production of analogs with enhanced antitumor activity. Predicted dioxygenases Orf12 and SibV associated with dihydropyrrole biosynthesis in PBDs anthramycin and sibiromycin, respectively, were expressed and purified for activity studies. UV-visible spectroscopy revealed that these enzymes catalyze the regiospecific 2,3-extradiol dioxygenation of l-3,4-dihydroxyphenylalanine (l-DOPA) to form l-2,3-secodopa (λmax=368 nm). (1)H NMR spectroscopy indicates that l-2,3-secodopa cyclizes into the α-keto acid tautomer of l-4-(2-oxo-3-butenoic-acid)-4,5-dihydropyrrole-2-carboxylic acid (λmax=414 nm). Thus, the dioxygenases are key for establishing the scaffold of the dihydropyrrole moiety. Kinetic studies suggest the dioxygenase product is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. The enzymatic product and dimeric state of these dioxygenases are conserved in dioxygenases involved in dihydropyrrole and pyrrolidine biosynthesis within both PBD and non-PBD pathways.

Sequence analysis of porothramycin biosynthetic gene cluster.

The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.

Molecular recognition of DNA by ligands: roughness and complexity of the free energy profile.

Understanding the molecular mechanism by which probes and chemotherapeutic agents bind to nucleic acids is a fundamental issue in modern drug design. From a computational perspective, valuable insights are gained by the estimation of free energy landscapes as a function of some collective variables (CVs), which are associated with the molecular recognition event. Unfortunately the choice of CVs is highly non-trivial because of DNA's high flexibility and the presence of multiple association-dissociation events at different locations and/or sliding within the grooves. Here we have applied a modified version of Locally-Scaled Diffusion Map (LSDMap), a nonlinear dimensionality reduction technique for decoupling multiple-timescale dynamics in macromolecular systems, to a metadynamics-based free energy landscape calculated using a set of intuitive CVs. We investigated the binding of the organic drug anthramycin to a DNA 14-mer duplex. By performing an extensive set of metadynamics simulations, we observed sliding of anthramycin along the full-length DNA minor groove, as well as several detachments from multiple sites, including the one identified by X-ray crystallography. As in the case of equilibrium processes, the LSDMap analysis is able to extract the most relevant collective motions, which are associated with the slow processes within the system, i.e., ligand diffusion along the minor groove and dissociation from it. Thus, LSDMap in combination with metadynamics (and possibly every equivalent method) emerges as a powerful method to describe the energetics of ligand binding to DNA without resorting to intuitive ad hoc reaction coordinates.

Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines.

Pyrrolobenzodiazepines (PBDs) are sequence selective DNA alkylating agents with remarkable antineoplastic activity. They are either naturally produced by actinomycetes or synthetically produced. The remarkable broad spectrum of activities of the naturally produced PBDs encouraged the synthesis of several PBDs, including dimeric and hybrid PBDs yielding to an improvement in the DNA-binding sequence specificity and in the potency of this class of compounds. However, limitation in the chemical synthesis prevented the testing of one of the most potent PBDs, sibiromycin, a naturally produced glycosylated PBDs. Only recently, the biosynthetic gene clusters for PBDs have been identified opening the doors to the production of glycosylated PBDs by mutasynthesis and biosynthetic engineering. This review describes the recent studies on the biosynthesis of naturally produced pyrrolobenzodiazepines. In addition, it provides an overview on the isolation and characterization of naturally produced PBDs, chemical synthesis of PBDs, mechanism of DNA alkylation, and DNA-binding affinity and cytotoxic properties of both naturally produced and synthetic pyrrolobenzodiazepines.

Usabamycins A-C: new anthramycin-type analogues from a marine-derived actinomycete.

New anthramycin-type analogues, designated usabamycin A-C (1, 2 and 3), have been isolated from cultures of Streptomyces sp. NPS853, a bacterium found in marine sediments. The structures of the new compounds were established on the basis of extensive spectroscopic analyses including 1D- and 2D-NMR ((1)H-(1)H COSY, HSQC, and HMBC) experiments. The usabamycins show weak inhibition of HeLa cell growth and selective inhibition of serotonin (5-hydroxytrypamine) 5-HT(2B) uptake.

A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin.

We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.

Concise formal synthesis of porothramycins A and B via Zincke pyridinium ring-opening/ring-closing cascade.

Short formal syntheses of the antitumor antibiotics porothramycins A and B from a commercially available ester of the unnatural amino acid 3-(3-pyridyl)alanine are presented. A rearrangement cascade that presumably involves a Zincke-type pyridinium ring-opening followed by cyclization of a pendant nucleophilic amide generates the salient pyrroline ring of the alkaloids.

Adenylation enzyme characterization using gamma -(18)O(4)-ATP pyrophosphate exchange.

We present here a rapid, highly sensitive nonradioactive assay for adenylation enzyme selectivity determination and characterization. This method measures the isotopic back exchange of unlabeled pyrophosphate into gamma-(18)O(4)-labeled ATP via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS), electrospray ionization liquid chromatography MS, or electrospray ionization liquid chromatography-tandem MS and is demonstrated for both nonribosomal (TycA, ValA) and ribosomal synthetases (TrpRS, LysRS) of known specificity. This low-volume (6 microl) method detects as little as 0.01% (600 fmol) exchange, comparable in sensitivity to previously reported radioactive assays and readily adaptable to kinetics measurements and high throughput analysis of a wide spectrum of synthetases. Finally, a previously uncharacterized A-T didomain from anthramycin biosynthesis in the thermophile S. refuinius was demonstrated to selectively activate 4-methyl-3-hydroxyanthranilic acid at 47 degrees C, providing biochemical evidence for a new aromatic beta-amino acid activating adenylation domain and the first functional analysis of the anthramycin biosynthetic gene cluster.

Solution structure of a 2:1 C2-(2-naphthyl) pyrrolo[2,1-c][1,4]benzodiazepine DNA adduct: molecular basis for unexpectedly high DNA helix stabilization.

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.

Dissociation of minor groove binders from DNA: insights from metadynamics simulations.

We have used metadynamics to investigate the mechanism of noncovalent dissociation from DNA by two representatives of alkylating and noncovalent minor groove (MG) binders. The compounds are anthramycin in its anhydrous form (IMI) and distamycin A (DST), which differ in mode of binding, size, flexibility and net charge. This choice enables to evaluate the influence of such factors on the mechanism of dissociation. Dissociation of IMI requires an activation free energy of approximately 12 kcal/mol and occurs via local widening of the MG and loss of contacts between the drug and one DNA strand, along with the insertion of waters in between. The detachment of DST occurs at a larger free energy cost, approximately 16.5 or approximately 18 kcal/mol depending on the binding mode. These values compare well with that of 16.6 kcal/mol extracted from stopped-flow experiments. In contrast to IMI, an intermediate is found in which the ligand is anchored to the DNA through its amidinium tail. From this conformation, binding and unbinding occur almost at the same rate. Comparison between DST and with kinetic models for the dissociation of Hoechst 33258 from DNA uncovers common characteristics across different classes of noncovalent MG ligands.

Reassembly of anthramycin biosynthetic gene cluster by using recombinogenic cassettes.

The reassembly and heterologous expression of complete gene clusters in shuttle vectors has enabled investigations of several large biosynthetic pathways in recent years. With a gene cluster in a mobile construct, the interrogation of gene functions from both culturable and nonculturable organisms is greatly accelerated and large pathway engineering efforts can be executed to produce "new" natural products. However, the genetic manipulation of complete natural product biosynthetic gene clusters is often complicated by their sheer size (10-200 kbp), which makes standard restriction/ligation-based methods impracticable. To circumvent these problems, alternative recombinogenic methods, which depend on engineered homology-based recombination have recently arisen as a powerful alternative. Here, we describe a new general technique that can be used to reconstruct large biosynthetic pathways from overlapping cosmids by retrofitting each cosmid with a "recombinogenic cassette" that contains a shared homologous element and orthogonal antibiotic markers. We employed this technique to reconstruct the anthramycin biosynthetic gene cluster of the thermotolerant actinomycete Streptomyces refuineus, from two >30 kbp cosmids into a single cosmid and integrate it into the genome of Streptomyces lividans. Anthramycin production in the heterologous Streptomyces host confirmed the integrity of the reconstructed pathway and validated the proposed boundaries of the gene cluster. Notably, anthramycin production by recombinant S. lividans was seen only during growth at high temperature--a property also shown by the natural host. This work provides tools to engineer the anthramycin biosynthetic pathway and to explore the connection between anthramycin production and growth at elevated temperatures.

Benzodiazepine biosynthesis in Streptomyces refuineus.

Anthramycin is a benzodiazepine alkaloid with potent antitumor and antibiotic activity produced by the thermophilic actinomycete Streptomyces refuineus sbsp. thermotolerans. In this study, the complete 32.5 kb gene cluster for the biosynthesis of anthramycin was identified by using a genome-scanning approach, and cluster boundaries were estimated via comparative genomics. A lambda-RED-mediated gene-replacement system was developed to provide supporting evidence for critical biosynthetic genes and to validate the boundaries of the proposed anthramycin gene cluster. Sequence analysis reveals that the 25 open reading frame anthramycin cluster contains genes consistent with the biosynthesis of the two halves of anthramycin: 4 methyl-3-hydroxyanthranilic acid and a "dehydroproline acrylamide" moiety. These nonproteinogenic amino acid precursors are condensed by a two-module nonribosomal peptide synthetase (NRPS) terminated by a reductase domain, consistent with the final hemiaminal oxidation state of anthramycin.