Sequence-Selective Interaction of the Minor-Groove Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA:  Footprinting and Enzyme Inhibition Studies

Abstract

SJG-136 (3) is a novel pyrrolobenzodiazepine (PBD) dimer that is predicted from molecular models to bind in the minor groove of DNA and to form sequence-selective interstrand cross-links at 5'-Pu-GATC-Py-3' (Pu = purine; Py = pyrimidine) sites through covalent bonding between each PBD unit and guanines on opposing strands. Footprinting studies have confirmed that high-affinity adducts do form at 5'-G-GATC-C-3' sequences and that these can inhibit RNA polymerase in a sequence-selective manner. At higher concentrations of SJG-136, bands that migrate more slowly than one of the 5'-G-GATC-C-3' footprint sites show significantly reduced intensity, concomitant with the appearance of higher molecular weight material near the gel origin. This phenomenon is attributed to interstrand cross-linking at the 5'-G-GATC-C-3' site and is the first report of DNA footprinting being used to detect interstrand cross-linked adducts. The control dimer GD113 (4), of similar structure to SJG-136 but unable to cross-link DNA due to its C7/C7'-linkage rather than C8/C8'-linkage, neither produces footprints with the same DNA sequence nor blocks transcription at comparable concentrations. In addition to the two high-affinity 5'-G-GATC-C-3' footprints on the MS2 DNA sequence, other SJG-136 adducts of lower affinity are observed that can still block transcription but with lower efficiency. All these sites contain the 5'-GXXC-3' motif (where XX includes AG, TA, GC, CT, TT, GG, and TC) and represent less-favored cross-link sites. In time-course experiments, SJG-136 blocks transcription if incubated with a double-stranded DNA template before the transcription components are added; addition after transcription is initiated fails to elicit blockage. Single-strand ligation PCR studies on a sequence from the c-jun gene show that SJG-136 binds to 5'-GAAC-3'/5'-GTTC-3' (preferred) or 5'-GAGC-3'/5'-GCTC-3' sequences. Significantly, adducts are obtained at the same sequences following extraction of DNA from drug-treated K562 cells, confirming that the agent reaches the cellular genome and interacts with the DNA in a sequence-selective fashion. Finally, SJG-136 efficiently inhibits the action of restriction endonuclease BglII, which has a 5'-A-GATC-T-3' motif at its cleavage site.

From Anthramycin to Pyrrolobenzodiazepine (PBD)‐Containing Antibody–Drug Conjugates (ADCs)

“…The labels to the right of each gel correspond to the potential binding sites 66. B) T‐Stop assay based on the same DNA sequence, showing the effect of incubation time on the ability of SJG‐136 (at 1.0 μ m ) to inhibit transcription.…”

<i>In Vitro</i> Investigation of DNA Damage Induced by the DNA Cross-Linking Agents Oxaliplatin and Satraplatin in Lymphocytes of Colorectal Cancer Patients

“…The formation of DNA adducts causes distortion in the DNA structure, including unwinding and bending, and obstructs DNA replication and transcription, thereby resulting in an enhancement in cell death (apoptosis) in vitro and cell-cycle arrest in the G2 phase [3]. Interstrand crosslinking (ICL) based anticancer agents such as cisplatin, carboplatin, and mitomycin C (MMC) are used generally in the clinic and novel ICL cancer anticancer therapeutics continue to be developed [4,5]. The discovery and development of platinum complexes is one of the great success stories in the advancement of cancer chemotherapy because of the pronounced activity of cisplatin in the treatment of testicular and ovarian cancers [6].…”

Entwicklung Pyrrolobenzodiazepin(PBD)‐haltiger Antikörper‐Wirkstoff‐Konjugate (ADCs) ausgehend von Anthramycin

“…[66] Die Sequenzselektivität von SJG-136 gegenüber isolierter DNA P-markierter pBR322-DNA zu zeigen. [10] Wiedergabe mit Genehmigung aus Lit.…”

“…Um zu untersuchen, ob die biologische In-vitro-und Invivo-Aktivität [57] von SJG-136 auf die sequenzselektive Inhibierung der Transkription zurückzuführen ist, wurde ein TStop-Assay an einer 282-bp-DNA-Sequenz durchgeführt, um seinen Einfluss auf T7-RNA-Polymerase und den Transkriptionsabbruch [22,66] zu verstehen (Abbildung 9 B). Es wurden wichtige Stopstellen (T-Stops) gefunden, die anscheinend hauptsächlich mit 5'-GXXC-3'-Sequenzen assoziiert sind.…”