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Die Pyrrolo[2,1 ‐ c][1,4]benzodiazepine (PBDs) sind eine Familie sequenzselektiver Wirkstoffe, die an die kleine Furche der DNA binden und eine kovalente Aminalbindung zwischen der C11 ‐ Position und den C2 ‐ NH2 ‐ Gruppen der Guaninbasen bilden. Anthramycin ist das erste Beispiel eines PBD ‐ Monomers und wurde in den 1960er Jahren entdeckt. In den 1990er Jahren wurde das bekannteste PBD ‐ Dimer SJG ‐ 136 entwickelt und hat jetzt bereits die klinischen Studien der Phase II in Patienten mit Leukämie und Eierstockkrebs durchlaufen. Seit kurzem werden aus PBD ‐ Dimeranaloga und spezifisch an Tumorzellen bindenden Antikörpern Antikörper ‐ Wirkstoff ‐ Konjugate (ADCs) hergestellt. Von diesen befinden sich derzeit mehrere in klinischen Studien und viele andere in der präklinischen Entwicklung. Dieser Aufsatz zeigt die Entwicklung der PBDs ausgehend von Anthramycin über die ersten PBD ‐ Dimere bis hin zu PBD ‐ haltigen ADCs und behandelt sowohl die Struktur ‐ Wirkungs ‐ Beziehungen und die Biologie der PBDs als auch die Strategien für ihre Verwendung als Wirkstoffkomponente in ADCs. PBDs in ADCs: Pyrrolobenzodiazepine (PBDs; Beispiel siehe Bild) sind DNA ‐ interaktive Tumortherapeutika, die sequenzselektiv an die Guaninbasen der kleinen Furche der DNA binden. Sie sind stark zytotoxisch und werden als Wirkstoffkomponente in Antikörper ‐ Wirkstoff ‐ Konjugaten (ADCs) verwendet. Dieser Aufsatz beschreibt ihre Entwicklung, ausgehend von der Entdeckung des Naturstoffs Anthramycin bis hin zur Verwendung von PBD ‐ Dimer ‐ Wirkstoffen in ADCs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. For the PBD-CPI dimer UTA-6026 (1), our simulations correctly predicted its favoured binding site (i.e., 50-C(G)AATTA-30) as identified by DNA cleavage studies. However, for the PBD-CI molecule ('Compound 11', 3), we were unable to reconcile the results of our simulations with the reported preferred cross-linking sequence (50-ATTTTCC(G)-30). We found that the molecule is too short to span the five base pairs between the A and G bases as claimed, but should target instead a sequence such as 50-ATTTC(G)-30 with two less base pairs between the reacting G and A residues. Our simulation results for this hybrid dimer are also in accord with the very low interstrand cross-linking and in vitro cytotoxicity activities reported for it. Although a preferred cross-linking sequence was not reported for the third hybrid dimer ('27eS', 2), our simulations predict that it should span two base pairs between covalently reacting G and A bases (e.g., 50-GTAT(A)-30). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Therefore, we evaluated the interaction of eight PBD molecules of diverse structure with a range of parallel, antiparallel and mixed DNA quadruplexes using DNA Thermal Denaturation, Circular Dichroism and Molecular Dynamics Simulations. Those PBD molecules without large C8-substitutents had an insignificant affinity for the eight quadruplex types, although those with large π-system-containing C8-substituents (as with the compounds evaluated by Raju and co-workers) were found to interact to some extent. Our molecular dynamics simulations support the likelihood that molecules of this type, including those examined by Raju and co-workers, interact with quadruplex DNA through their C8-substituents rather than the PBD moiety itself. It is important for the literature to be clear on this matter, as the mechanism of action of these agents will be under close scrutiny in the near future due to the growing number of PBD-based agents entering the clinic as both single-agents and as components of antibody-drug conjugates (ADCs). [ABSTRACT FROM AUTHOR]

DNA binding 4-(1-methyl-1H-pyrrol-3-yl)benzenamine(MPB) building blocks have been developed that span two DNA base pairswith a strong preference for GC-rich DNA. They have been conjugatedto a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) moleculeto produce C8-linked PBD–MPB hybrids that can stabilize GC-richDNA by up to 13-fold compared to AT-rich DNA. Some have subpicomolarIC50values in human tumor cell lines and in primary chroniclymphocytic leukemia cells, while being up to 6 orders less cytotoxicin the non-tumor cell line WI38, suggesting that key DNA sequencesmay be relevant targets in these ultrasensitive cancer cell lines.One conjugate, 7h(KMR-28-39), which has femtomolar activityin the breast cancer cell line MDA-MB-231, has significant dose-dependentantitumor activity in MDA-MB-231 (breast) and MIA PaCa-2 (pancreatic)human tumor xenograft mouse models with insignificant toxicity attherapeutic doses. Preliminary studies suggest that 7hmay sterically inhibit interaction of the transcription factor NF-κBwith its cognate DNA binding sequence. [ABSTRACT FROM AUTHOR]

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. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo. [ABSTRACT FROM AUTHOR]

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. The development of new ADCs is challenging, particularly the process of attaching the linker-payload construct to the antibody (i.e., the conjugation process). One of the major problems associated with conjugation is high hydrophobicity of the payload which can lead to low yields of the ADC through aggregation and/or lower than desired Drug-Antibody Ratios (DARs). We report here a UPLC-based assay that can be used to study the physicochemical properties of ADC payloads at an early stage of development, and to provide information on whether the hydrophilic-hydrophobic balance is suitable for conjugation or further physicochemical optimization is required. The assay is relatively simple to establish and should be of use to those working in the ADC area., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: IP has share options and PJMJ, KMR and DET have founding stock in Femtogenix Ltd, the company supplying one of the experimental ADC payloads evaluated in this study. IP and PJMJ are fully employed by Femtogenix Ltd, and KMR and DET have consultancies with the company. PJMJ, KMR and DET are named inventors on patents filed and owned by the company., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018. Published by Elsevier Ltd.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of sequence-selective, DNA minor-groove binding agents that covalently attach to guanine residues. Originally derived from Streptomyces species, a number of naturally occurring PBD monomers exist with varying A-Ring and C2-substituents. One such agent, sibiromycin, is unusual in having a glycosyl residue (sibirosamine) at its A-Ring C7-position. It is the most cytotoxic member of the naturally occurring PBD family and has the highest DNA-binding affinity. Recently, the analogue 9-deoxysibiromyin was produced biosynthetically by Yonemoto and co-workers.1 Differing only in the loss of the A-Ring C9-hydroxyl group, it was reported to have a significantly higher DNA-binding affinity than sibiromycin based on DNA thermal denaturation studies, although these data have since been retracted.2 As deletion of the C9-OH moiety, which points toward the DNA minor groove floor, might intuitively be expected to reduce DNA-binding affinity through the loss of hydrogen bonding, we carried out molecular dynamics simulations on the interaction of both molecules with DNA over a 10 ns time-course in explicit solvent. Our results suggest that the two molecules may differ in their sequence-selectivity and that 9-deoxysibiromycin should have a lower binding affinity for certain sequences of DNA compared to sibiromycin. Our molecular dynamics results indicate that the C7-sibirosamine sugar does not form hydrogen bonding interactions with groups in the DNA minor-groove wall as previously reported, but instead points orthogonally out from the minor groove where it may inhibit the approach of DNA control proteins such as transcription factors. This was confirmed through a docking study involving sibiromycin and the GAL4 transcription factor, and these results could explain the significantly enhanced cytotoxicity of sibiromycin compared to other PBD family members without bulky C7-substituents.

The binding of nuclear factor Y (NF-Y) to inverted CCAAT boxes (ICBs) within the promoter region of DNA topoisomerase IIα results in control of cell differentiation and cell cycle progression. Thus, NF-Y inhibitory small molecules could be employed to inhibit the replication of cancer cells. A library of pyrrolobenzodiazepine (PBD) C8-conjugates consisting of one PBD unit attached to tri-heterocyclic polyamide fragments was designed and synthesized. The DNA-binding affinity and sequence selectivity of each compound were evaluated in DNA thermal denaturation and DNase I footprinting assays, and the ability to inhibit binding of NF-Y to ICB1 and ICB2 was studied using an electrophoretic mobility shift assay (EMSA). 3a was found to be a potent inhibitor of NF-Y binding, exhibiting a 10-fold selectivity for an ICB2 site compared to an ICB1-containing sequence, and showing low nanomolar cytotoxicity toward human tumor cell lines. Molecular modeling and computational studies have provided details of the covalent attachment process that leads to formation of the PBD-DNA adduct, and have allowed the preference of 3a for ICB2 to be rationalized.

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. Alkylation of four cysteine residues has been observed with possible reaction at a fifth which could account for the mechanism of action., (Copyright © 2013. Published by Elsevier Ltd.)