Structure of Pyrrolobenzodiazepine
Structure of Pyrrolobenzodiazepine

Pyrrolobenzodiazepine (PBD)
Chemical Name: [1,2]Diazepino[3,4-e]indole;
Molecular Formula: C14H14N2O3
Molecular weight: 258,27 g/mole
Average mass: 181.193 Da
Monoisotopic mass: 181.063995 Da

Introduction
Discovered in 1963, the pyrrolobenzodiazepines (PBDs) are a class of antitumor antibiotics which include the naturally occurring anthramycin, sibiromycin, tomaymycin, the neothramycin A and neothramycin B, and DC-81. Other PBD antibiotics include abbeymycin, chicamycin, mazethramycin, porothramycin, prothracarcin, sibanomicin (DC-102).

PBDs are sequence-selective DNA minor-groove binding crosslinking agents originally discovered in Streptomyces species and are significantly more potent than systemic chemotherapeutic drugs. Pyrrolobenzodiazepines are potent inhibitors of nucleic acid synthesis because of their ability to recognize and bind to specific sequences of DNA and form a labile covalent adduct. [1][2]

Although PBDs have high antitumor activity, they are cardiotoxic, which has precluded their continued clinical use.

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Ongoing research demonstrates that PBDs can be effectively used for antibody-targeted therapy. [3][4][5]

Biological Activity
The mechanism of action (MOA) of the PBDs is associated with their ability to form, an adduct in the minor groove, thus interfering with DNA processing. After insertion in the minor groove, an aminal bond is formed through the nucleophilic attack of the N2 of a guanine base at the electrophilic C11 position of PBD.[6]

PBD dimer mode of action. PBD dimers bind to specially configured guanidine residues on different positions of the dsDNA helix and cause the crosslinking of DNA strands (Adapted from Org. Biomol. Chem., 2015).
PBD dimer mode of action: PBD dimers bind to specially configured guanidine residues on different positions of the dsDNA helix and cause the crosslinking of DNA strands (Adapted from Org. Biomol. Chem., 2015).

What are Pyrrolobenzodiazepines?
Pyrrolobenzodiazepines, a class of natural products produced by various actinomycetes (a broad group of bacteria that form thread-like filaments in the soil and are responsible for the distinctive scent of freshly exposed, moist soil), are sequence selective DNA alkylating compounds with significant antitumor properties.

As a class of DNA-crosslinking agents, they are significantly more potent than systemic chemotherapeutic drugs. Some PBDs have the ability to recognize and bond to specific sequences of DNA.[7][9]

PBD dimers bind in the minor groove of DNA, where they form covalent aminal cross-links between the N2 of guanine and the C11 position of the PBD. The resulting PBD-DNA adducts cause replication forks to stall and tumor cells to arrest at the G2-M boundary, ultimately resulting in apoptosis at low nanomolar to picomolar concentrations. PBD dimers are particularly potent because of their cell cycle–independent activity and because their integration minimally distorts DNA, increasing the likelihood of evasion of DNA damage repair responses. Because of the potency of PBD dimers and similar cell cycle–independent payloads, normal tissues accessed by such potently armed ADCs must be devoid of target expression.[8]

As DNA minor groove binding agents, pyrrolobenzodiazepines bind and cross-link specific sites of DNA of the cancer cell. This blocks the cancer cells’ division without distorting its DNA helix, thus potentially avoiding the common phenomenon of emergent drug resistance.

Skeletal formula of anthramycin—a pyrrolobenzodiazepine antibiotic and antineoplastic.
Figure 2.0: Skeletal formula of anthramycin—a pyrrolobenzodiazepine antibiotic and antineoplastic.

Anthramycin
The first pyrrolobenzodiazepines antitumor antibiotic, anthramycin (C16H17N3O4; not to be confused with anthracimycin), produced by streptomyces refuineus, was discovered in 1965.

Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogs.[9]

Potency
In vitro, pyrrolobenzodiazepines typically demonstrate IC50 values in the low to mid picomolar range in a variety of cell types, and unlike the anti-tubulin agents, they can induce cell death in both dividing and non-dividing cells. Fully synthetic PBD dimers are ideally suited for the role of payload in an antibody-drug conjugate because, unlike other cytotoxic payloads such as calicheamicin, they combine potency with a demonstrated therapeutic index, are not cross-resistant with widely used chemotherapy agents, and their unique mode of action sets them apart from the tubulin binders such as maytansinoid and auristatin that currently dominate the antibody-drug conjugate arena.

The pyrrolobenzodiazepine (PBD) dimers are significantly more potent than systemic chemotherapeutic drugs and the site-specific conjugation technology allows uniform drug-loading of the cell-killing PBD agent to the anti-CD33 antibody. Pyrrolobenzodiazepine dimers have been shown to have broad-spectrum anti-tumor activity in vivo. These novel drugs exert their activity by binding in the minor groove of DNA and linking the two DNA strands together in a way that cells find difficult to recognize and repair.

Binding in the minor groove of DNA
PBDs exert their biological activity by binding in the minor groove of DNA with selectivity for 5′-purine-guanine-purine sequences and forming a covalent bond to the exocyclic amino group of the guanine base.

Synthetic PBD dimers – which links two PBD monomers through their aromatic A-ring phenolic C8-positions via a flexible propyldioxy tether, have been found to be highly cytotoxic in vitro.  These synthetic PBD dimers produce DNA interstrand cross-links with high efficiency in both naked DNA and in cells. These types of molecules span six base pairs in the minor groove of DNA, covalently binding to spatially separated guanines on opposite strands in the central sequence 5′-PuGATCPy-3′ (where Pu is a purine and Py is a pyrimidine). Using HPLC/MS and short oligonucleotides of the defined sequence also suggest that PBD dimers form sequence selective intrastrand cross-linked adducts and mono-alkylated adducts, in addition to interstrand cross-links.

One observed and important feature of PBD dimers and the interstrand cross-links formed is that they cause minimal distortion of the DNA. This feature appears to contribute to the persistence in cells and the potent biological activity of these agents.

Development
Spirogen, a wholly-owned subsidiary of AstraZeneca’s MedImmune founded in 2001 as a spin-out from several institutions including University College London and with partial funding by Cancer Research UK, has developed a novel class of highly potent cytotoxic warheads based on its proprietary pyrrolobenzodiazepines (PBDs). The company has been developing its PBD technology for more than ten years, including a standalone PBD agent in a Phase II study in acute myeloid leukemia.

Other companies involved in the development of ADCs with a PBD payload include Seattle Genetics and ADC Therapeutics.


Pyrrolobenzodiazepine_DimerPyrrolobenzodiazepine Dimer
Molecular Formula: C31H32N4O6
Molecular Weight: 556,61 g/mole

See: ADC Review / Knowledge Center 


Clinical development with Pyrrolobenzodiazepine (PBD)

AbbVie/Stemcentrx 

  • Rovalpituzumab tesirine (Rova-T | SC16LD6.5 | DLL-3 targeted ADC).  In August 2019, AbbVie Discontinued the Rovalpituzumab Tesirine Research and Development Program.

Seattle Genetics

  • Vadastuximab Talirine (SGN-CD33A): a CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML. In June 2017 Seattle Genetics stopped all clinical testing of vadastuximab talirine (SGN-CD33A) after seeing a higher rate of patient deaths with the drug in a phase III trial. Several earlier-stage trials of the CD33-targeting antibody-drug conjugate were halted by the U.S. FDA in December 2016 on safety concerns, primarily relating to the possibility of liver toxicities.
  • SGN-CD70a

ADC Therapeutics


Reference:
[1] Leimgruber W, Stefanović V, Schenker F, Karr A, Berger J. Isolation and characterization of anthramycin, a new antitumor antibiotic. J Am Chem Soc. 1965;87(24):5791‐5793. doi:10.1021/ja00952a050
[2] Tendler MD, Korman S. ‘Refuin’: A Non-Cytotoxic Carcinostatic Compound Proliferated by a Thermophilic Acinomycete Nature. 1963;199:501. doi:10.1038/199501a0
[3] Antonow D, Thurston DE (2011) Synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines (PBDs). Chem Rev 111: 2815–2864. doi: 10.1021/cr100120f
[4] Cipolla L, Araujo AC, Airoldi C, Bini D (2009) Pyrrolo[2,1-c][1,4]benzodiazepine as a scaffold for the design and synthesis of anti-tumor drugs. Anticancer Agents Med Chem 9: 1–31. doi: 10.2174/187152009787047743
[5] Gerratana B (2012) Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines. Med Res Rev 32: 254–293. doi: 10.1002/med.20212
[6] Li W, Khullar A, Chou S, Sacramo A, Gerratana B. Biosynthesis of sibiromycin, a potent antitumor antibiotic. Appl Environ Microbiol. 2009 May;75(9):2869-78. doi: 10.1128/AEM.02326-08. Epub 2009 Mar 6.
[7] Rahman KM, Jamesa CH, Thurston DE. Observation of the reversibility of a covalent pyrrolobenzodiazepine (PBD) DNA adduct by HPLC/MS and CD spectroscopy. Org. Biomol. Chem., 2011,9, 1632-1641. DOI: 10.1039/C0OB00762E
[8] Saunders LR, Bankovich AJ, Anderson WC, Aujay MA, Bheddah S, Black K, Desai R, et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med. 2015 Aug 26;7(302):302ra136. doi: 10.1126/scitranslmed.aac9459.
[9] Hu Y, Phelan V, Ntai I, Farnet CM, Zazopoulos E, Bachmann BO. Benzodiazepine biosynthesis in Streptomyces refuineus. Chem Biol. 2007 Jun;14(6):691-701.
[10] Hartley JA, Flynn MJ, Bingham JP, et al. Pre-clinical pharmacology and mechanism of action of SG3199, the pyrrolobenzodiazepine (PBD) dimer warhead component of antibody-drug conjugate (ADC) payload tesirine. Sci Rep. 2018;8(1):10479. Published 2018 Jul 11. doi:10.1038/s41598-018-28533-4