DNA sequence-selective G-A cross-linking ADC payloads for use in solid tumour therapies.

Antibody-Drug Conjugates (ADCs) are growing in importance for the treatment of both solid and haematological malignancies. There is a demand for new payloads with novel mechanisms of action that may offer enhanced therapeutic efficacy, especially in patients who develop resistance. We report here a class of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) DNA cross-linking payloads that simultaneously alkylate guanine (G) and adenine (A) bases in the DNA minor groove with a defined sequence selectivity.

UPLC-based assay to assess the hydrophobicity of Antibody-Drug Conjugate (ADC) payloads.

Antibody-Drug Conjugates (ADCs) consist of antibodies attached to cytotoxic small molecules or biological agents (i.e., payloads) through chemical linkers which may be cleavable or non-cleavable.

A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma.

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class.

Use of pyrrolobenzodiazepines and related covalent-binding DNA-interactive molecules as ADC payloads: Is mechanism related to systemic toxicity?

Antibody-drug conjugates (ADCs) consist of monoclonal antibodies (mAbs) or antibody fragments conjugated to biologically active molecules (usually highly cytotoxic small molecules) through chemical linkers. Although no ADCs containing covalent-binding DNA-interactive payloads have yet been approved (although two containing the DNA-cleaving payload calicheamicin have), of those in clinical trials systemic toxicities are beginning to emerge. This article discusses the observed toxicities in relation to the structures and mechanisms of action of payload type.

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.

The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G-A cross-linking PBD-duocarmycin dimers.

The pyrrolobenzodiazepine (PBD) and duocarmycin families are DNA-interactive agents that covalently bond to guanine (G) and adenine (A) bases, respectively, and that have been joined together to create synthetic dimers capable of cross-linking G-G, A-A, and G-A bases. Three G-A alkylating dimers have been reported in publications to date, with defined DNA-binding sites proposed for two of them. In this study we have used molecular dynamics simulations to elucidate preferred DNA-binding sites for the three published molecular types.

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-NH 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.

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.

Effect of hairpin loop structure on reactivity, sequence preference and adduct orientation of a DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumour agent.

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.

Crispene E, a cis-clerodane diterpene inhibits STAT3 dimerization in breast cancer cells.

Crispene E, a new clerodane-type diterpene, inhibited STAT3 dimerization in a cell-free fluorescent polarisation assay and was found to have significant toxicity against STAT3-dependent MDA-MB 231 breast cancer cell line and selectively inhibited the expression of STAT3 and STAT3 target genes cyclin D1, Fascin and bcl-2. Molecular docking studies suggest the molecule inhibits STAT3 by interacting with its SH2 domain. The compound has been isolated from Tinospora crispa and characterized using standard spectroscopic techniques.

Pyrrolobenzodiazepines (PBDs) do not bind to DNA G-quadruplexes.

The pyrrolo[2,1-c][1,4] benzodiazepines (PBDs) are a family of sequence-selective, minor-groove binding DNA-interactive agents that covalently attach to guanine residues. A recent publication in this journal (Raju et al, PloS One, 2012, 7, 4, e35920) reported that two PBD molecules were observed to bind with high affinity to the telomeric quadruplex of Tetrahymena glaucoma based on Electrospray Ionisation Mass Spectrometry (ESI-MS), Circular Dichroism, UV-Visible and Fluorescence spectroscopy data. This was a surprising result given the close 3-dimensional shape match between the structure of all PBD molecules and the minor groove of duplex DNA, and the completely different 3-dimensional structure of quadruplex DNA.

Computational studies support the role of the C7-sibirosamine sugar of the pyrrolobenzodiazepine (PBD) sibiromycin in transcription factor inhibition.

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.

An extended pyrrolobenzodiazepine-polyamide conjugate with selectivity for a DNA sequence containing the ICB2 transcription factor binding site.

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.

Investigation of the protein alkylation sites of the STAT3:STAT3 inhibitor Stattic by mass spectrometry.

STAT3 (Signal Transducer and Activator of Transcription factor 3) is constitutively active in a wide range of human tumours. Stattic is one of the first non-peptidic small molecules reported to inhibit formation of the STAT3:STAT3 protein dimer complex. A mass spectrometry method has been developed to investigate the binding of Stattic to the un-phosphorylated STAT3βtc (U-STAT3) protein.

GC-targeted C8-linked pyrrolobenzodiazepine-biaryl conjugates with femtomolar in vitro cytotoxicity and in vivo antitumor activity in mouse models.

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.