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.

Rapid conjugation of antibodies to toxins to select candidates for the development of anticancer Antibody-Drug Conjugates (ADCs).

Antibody-Drug Conjugates (ADCs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with eight ADCs and two immunotoxins approved for clinical use. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation.

Novel pyrrolobenzodiazepine benzofused hybrid molecules inhibit NF-κB activity and synergise with bortezomib and ibrutinib in hematological cancers.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are incurable hematological malignancies that are pathologically linked with aberrant NF-κB activation. In this study, we identified a group of novel C8-linked benzofused Pyrrolo[2,1-c][1,4]benzodiazepines (PBD) monomeric hybrids capable of sequence-selective inhibition of NF-κB with low nanomolar LD50 values in CLL (n=46) and MM cell lines (n=5). The lead compound, DC-1-192, significantly inhibited NF-κB DNA binding after just 4h exposure and demonstrating inhibitory effects on both canonical and non-canonical NF-κB subunits.

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.

Effects of Systematic Shortening of Noncovalent C8 Side Chain on the Cytotoxicity and NF-κB Inhibitory Capacity of Pyrrolobenzodiazepines (PBDs).

The systematic shortening of the noncovalent element of a C8-linked pyrrolobenzodiazepine (PBD) conjugate (13) led to the synthesis of a 19-member library of C8-PBD monomers. The critical elements of 13, which were required to render the molecule cytotoxic, were elucidated by an annexin V assay. The effects of shortening the noncovalent element of the molecule on transcription factor inhibitory capacity were also explored through an enzyme-linked immunosorbent assay-based measurement of nuclear NF-κB upon exposure of JJN-3 cells to the synthesized molecules.

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.

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).

Antibody structure and engineering considerations for the design and function of Antibody Drug Conjugates (ADCs).

Antibody-drug conjugates (ADCs) are emerging as effective tools in cancer therapy, combining the antibody's exquisite specificity for the target antigen-expressing cancer cell together with the cytotoxic potency of the payload. Much success stems from the rational design of "toxic warheads", chemically linked to antibodies, and from fine-tuning the intricate properties of chemical linkers. Here, we focus on the antibody moiety of ADCs, dissecting the impact of Fab, linkers, isotype and Fc structure on the anti-tumoral and immune-activating functions of ADCs.

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.

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.

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.

Recent advances in targeting the telomeric G-quadruplex DNA sequence with small molecules as a strategy for anticancer therapies.

Human telomeric DNA (hTelo), present at the ends of chromosomes to protect their integrity during cell division, comprises tandem repeats of the sequence d(TTAGGG) which is known to form a G-quadruplex secondary structure. This unique structural formation of DNA is distinct from the well-known helical structure that most genomic DNA is thought to adopt, and has recently gained prominence as a molecular target for new types of anticancer agents. In particular, compounds that can stabilize the intramolecular G-quadruplex formed within the human telomeric DNA sequence can inhibit the activity of the enzyme telomerase which is known to be upregulated in tumor cells and is a major contributor to their immortality.

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.

Activity of the DNA minor groove cross-linking agent SG2000 (SJG-136) against canine tumours.

Background: Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent.

Topical therapies for skin cancer and actinic keratosis.

The global incidence of skin cancer and actinic keratosis (AK) has increased dramatically in recent years. Although many tumours are treated with surgery or radiotherapy topical therapy has a place in the management of certain superficial skin neoplasms and AK. This review considers skin physiology, non-melanoma skin cancer (NMSC), the relationship between AK and skin cancer and drugs administered topically for these conditions.

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.

Novel 1-(2-aryl-2-adamantyl)piperazine derivatives with antiproliferative activity.

Novel 1-(2-aryl-2-adamantyl)piperazine derivatives have been synthesized and evaluated in vitro for their antitumor properties against HeLa cervical carcinoma, MDA MB 231 breast cancer, MIA PaCa2 pancreatic cancer, and NCI H1975 non-small cell lung cancer. The parent piperazine 6 was found to exhibit a reasonable activity toward the HeLa and MDA MB 231 tumor cell lines (IC50= 9.2 and 8.

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.