SG2285, a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity.

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.

An asymmetric C8/C8'-tripyrrole-linked sequence-selective pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer DNA interstrand cross-linking agent spanning 11 DNA base pairs.

A novel sequence-selective extended PBD dimer 4 has been synthesized that binds with high affinity to an interstrand cross-linking site spanning 11 DNA base pairs. Despite its molecular weight (984.07) and length, the molecule has significant DNA interstrand cross-linking potency (approximately 100-fold greater than the clinically used agent melphalan) and sub-micromolar cytotoxicity in a number of tumour cell lines, suggesting that it readily penetrates cellular and nuclear membranes to reach its DNA target.

A 96-well DNase I footprinting screen for drug-DNA interactions.

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive 'one compound-one line' scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.

Design, synthesis, and biophysical and biological evaluation of a series of pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugates.

A novel series of methyl ester-terminated C8-linked pyrrolobenzodiazepine (PBD)-poly(N-methylpyrrole) conjugates (50a-f) has been synthesized and their DNA interaction evaluated by thermal denaturation, DNA footprinting, and in vitro transcription stop assays. The synergistic effect of attaching a PBD unit to a polypyrrole fragment is illustrated by the large increase in DNA binding affinity (up to 50-fold) compared to the individual PBD and pyrrole components. 50a-f were found to bind mainly to identical DNA sequences but with apparent binding site widths increasing with molecular length and the majority of sites conforming to the consensus motif 5'-XGXWz (z = 3 +/- 1; W = A or T; X = any base but preferably a purine). They also provided robust sequence-selective blockade of transcription at sites corresponding approximately to their DNA footprints. 50a-f were shown to have good cellular/nuclear penetration properties, and a degree of correlation between cytotoxicity and DNA-binding affinity was observed.

Unusually strong positive cooperativity in binding of peptides to latent membrane protein-1 DNA fragments of the Epstein-Barr viral gene.

The DNA-binding preferences of two oligopeptide amides, (His-Pro-Arg-Lys)(3)NH(2) (HR-12) and (Ser-Pro-Arg-Lys)(3)NH(2) (SP-12), have been examined by quantitative DNase I footprinting studies. Two different DNA fragments were investigated: a pair of 5'-(32)P-labeled duplexes from pBR322 with one or other of the complementary strands labeled and a corresponding pair of 5'-(32)P-labeled duplexes representing fragments of the latent membrane protein (LMP-1) gene from a pathogenic Epstein-Barr virus variant derived from nasopharyngeal carcinoma. The major objective was to examine molecular recognition and cooperative features associated with sequence-selective binding of synthetic peptides to the LMP-1 fragments. At various binding sites on the pBR322 fragments, Hill coefficients (n(H)) ranging from 1.9-2.2 were observed; these results indicate modest positive cooperativity between binding sites for both peptides. By contrast, unusually high values of n(H), ranging from 4.0-9.3, were observed at various binding sites on the LMP-1 fragments. Allosteric models can be constructed to interpret the observed cooperative interactions between different DNA recognition sites in the LMP-1 gene upon binding of the peptide ligands. It is noteworthy that these models feature a novel network of cooperativity interconnecting multiple DNA allosteric sites. The evidence of sequence selectivity and strong cooperativity discovered in this work may prove to be a general feature of peptide interactions with some nucleic acids.

Preferential binding to DNA sequences of peptides related to a novel XPRK motif.

Two dodecapeptide amines: (WPRK)(3)NH(2)[WR-12] and (YPRK)(3)NH(2)[YR-12], and a 30-mer polypeptide amide (SP-30) were synthesized by solid-phase peptide methodology. DNase I footprinting studies on a 117-mer DNA showed that WR-12 and YR-12 bind selectively to DNA sequences in a manner similar to SP-30 which has a repeating SPK(R)K sequence. The most distinctive blockages seen with all three peptides occur at positions 26-30, 21-24 and 38-45 around sequences 5'-GAATT-3', 5'-TAAT-3' and 5'-AAAACGAC-3', respectively. However, it appears that YR-12 is better able to extend its recognition site to include CG pairs than is SP-30. At low concentrations YR-12 was able to induce enhanced rates of DNase I cleavage at regions surrounding some of its binding sites. To obtain further quantitative data supplementary to the footprinting work, equilibrium binding experiments were performed in which the binding of the two peptides to six decanucleotide duplexes was compared. Scatchard analyses indicated that WR-12 may be more selective for oligomers containing runs of consecutive purines or pyrimidines. On the other hand, YR-12 binds better to d(CTTAGACGTC)- d(GACGTCTAAG) than to the other oligomer duplexes, denoting selectivity for evenly distributed C/G and A/T sequences.