The Pyrrolobenzodiazepine Dimer SJG-136 Forms Sequence-Dependent Intrastrand DNA Cross-Links and Monoalkylated Adducts in Addition to Interstrand Cross-Links

Abstract

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials. Using a HPLC/MS-based methodology developed to evaluate the binding of DNA-interactive agents to oligonucleotides of varying length and sequence, we have demonstrated that, in addition to the previously known interstrand cross-link at Pu-GATC-Py sequences, 1 can form a longer interstrand cross-link at Pu-GAATC-Py sequences, an intrastrand cross-link at both shorter Pu-GATG-Py and longer Pu-GAATG-Py sequences, and, in addition, monoalkylated adducts at suitable PBD binding sites where neither intra- or interstrand cross-links are feasible because of the unavailability of two appropriately positioned guanines. Crucially, we have demonstrated a preference for the extended intrastrand cross-link with Pu-GAATG-Py, which forms more rapidly than the other cross-links (rank order: Pu-GAATG-Py > Pu-GATC-Py >> Pu-GATG-Py and Pu-GAATC-Py). However, thermal denaturation studies suggest that the originally reported Pu-GATC-Py interstrand cross-link is more stable, consistent with the covalent joining of both strands of the duplex and a lower overall distortion of the helix according to modeling studies. These observations impact on the proposed mechanism of action of SJG-136 (1) both in vitro and in vivo, the repair of its adducts and mechanism of resistance in cells, and potentially on the type of pharmacodynamic assay used in clinical trials.

DNA Sequence Preference and Adduct Orientation of Pyrrolo[2,1-c][1,4]benzodiazepine Antitumor Agents

“…However, a similar model has been proposed for the interaction of one PBD unit of the PBD dimer SJG-136 with DNAwhen forming an intrastrand cross-link. 15 Finally, the results of the free energy calculations ( Table 1), which relate to the thermodynamic stability of the PBD-DNA adducts, are in broad agreement with literature reports that the preferred orientation of a PBD adduct is with the A ring pointing toward the 3 0 -end of the covalently modified DNA strand. For example, between pairs of oligonucleotides with AGA (Seq-1 and Seq-5), TGT (Seq-2 and Seq-6), AGT (Seq-3 and Seq-7), and TGA (Seq-4 and Seq-8) sequences, lower energies were obtained for the 3 0 -orientation (i.e., for Seq-5, Seq-6, Seq-7, and Seq-8) in each case.…”

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

“…[11] Die C2/C8'-verknüpften PBD-Dimere, die mithilfe einer Klick-Chemie-Methode erhalten wurden, banden ähnlich schlecht an DNA. [12] Die einzigartige Struktur der PBD-Dimere, die zwei alkylierende Iminfunktionen enthalten, ermçglicht ihnen zusätzlich zur Bildung von monoalkylierten Addukten auch die Bildung von Vernetzungen zwischen DNA-Strängen oder innerhalb eines DNA-Strangs, [14] was zu einer besseren DNA-Stabilisie- Abbildung 2. A) Der Mechanismus der kovalenten Anbindung eines PBD an DNA, sobald es in einer niederenergetischen Position in der kleinen Furche lokalisiert ist.…”

“…Im zweiten Schritt bildet das PBD eine oder zwei kovalente Bindungen mit der oder den Guaninbase(n), und das Molekül ist dann in dieser Position fest gebunden. Wäh-rend die anfängliche nichtkovalente Assoziation rasch verläuft, ist der Schritt der kovalenten Anbindung je nach PBDStruktur und DNA-Sequenz verschieden schnell und kann zwischen 3 h [4] und 24 h [14] dauern (Abbildung 2 A). Wenn sie erst einmal an DNA gebunden sind, kçnnen PBD-Monomere und -Dimere eine Reihe biologischer Effekte in Zellen haben.…”

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

“…Through the years, many novel cross-linking agents have been designed, synthesized and evaluated [4] , [5] in an attempt to (a) improve on cross-linking efficiency [6], (b) modify the sequence-selectivity of cross-linking [7], (c) change the distribution of inter-versus intrastrand cross-links [8][9][10], and/or (d) target different base pair combinations [11][12][13]. For example, the naturally occurring pyrrolobenzodiazepine (PBD) molecules, derived from Streptomyces species [14][15][16][17][18], are characterized by an N10-C11 imine group that enables formation of a covalent bond to the C2-amino group of a guanine base [19], and these have been successfully incorporated into PBD dimer structures such as DSB-120 [20] and SJG-136 [21] (Figure 1A) that can form sequence-selective G-G cross-links in the DNA minor groove.…”

“…The cytotoxicity of these molecules is thought to be directly related to the formation of interstrand cross-links (in this instance at Pu-GATC-Py sites [22,23]), and members of the PBD dimer class are now being used as payloads for antibody-drug conjugates (ADCs) [24,25]. Evidence is accumulating to suggest additional mechanisms of action including the formation of intrastrand cross-links [9,10,26] and mono-adducts, the inhibition of transcription factor binding [27,28] and the inhibition of enzymes such as endonucleases [29,30] and RNA polymerase [31].…”